In 1424, Mt Athos finally fell under Ottoman control. At first the new political authority recognized the existing ownership rights and the associated benefits of the monasteries. However, they soon levied taxes not only on the monasteries on Mt Athos, but also on the Athonite dependencies located elsewhere, many of which were lost as a result, while the arbitrary behaviour of tax officials and other state employees was a common problem. Consequently, the economic condition of the monasteries greatly worsened, the number of monks decreased, and in general the functional bonds between the Athonite monasteries and the world outside it were greatly weakened. The amount of tax due from the Karakallou Monastery is not known precisely, i.e. what percentage of the total taxes levied on the Mt Athos monasteries had been apportioned to it, but based on an Ottoman inventory record of 1520, it was probably one of the few surviving monasteries on the peninsula at that time. If they had succeeded, as all the monasteries had sought to do, in not being taxed based on a system of tithes on agricultural production and other related analogous taxes, but instead based on the more beneficial system of a flat tax rate, that would have meant that the amount of tax would have remained stable for a long time and would have reduced the chance of arbitrarily imposed taxes.
There are few reports about the Monastery during the first decades after the Ottoman conquest. In 1444, the Monastery was visited by the Italian traveller Kyriakos Agonitis, who writes that he met the Prior David and poor Serbian monks there who, even though they spoke a foreign language, served the Greek Orthodox church. This report about the presence of Serbian monks at the Monastery in the first half of the 15th century is unique. Moreover, it is noteworthy because until that time, the Monastery clearly had a Greek character, with evidence of the last known Prior, Gregorios, having signed his name in Greek in 1423. The next known testimony about the Monastery dates to 1486/87, and comes from a document of the Proto Kosmas which delineates the border between the Filotheos and Karakallou monasteries, an event at which the Prior Iakobos of Karakallou Monastery was present. A copy of this document from the 17th or 18th century survives, but we do not know whether the original was written in Greek or in Slavic.
The next known archived testimonies referring to representatives of the Monastery are the signatures of the Prior Stefanos in 1503 on a document issued by the Proto, and that of the Prior Maximos in 1504/05 on a document by the Proto Moyseos. Both signatures were in Slavic. These seem to be the last pieces of evidence of their kind: There are numerous pieces of evidences from 1518 which bear signatures of the representatives of the Monastery, and all are in Greek. Thus, there may be a basis for the hypothesis that for a short period of time, shortly before the middle of the 15th century and until the beginning of the 16th, the Monastery was inhabited by non-Greek-speaking monks, as was the case in other Athonite monasteries as well.
In the 16th century, Karakallou was organized according to the idiorhythmic system of monastic life. The office of Abbot was not discontinued, but instead took on a mainly ritual character, being awarded to those who performed outstanding services to the Monastery (e.g. successfully carrying out something requested, sound management of a dependency, etc.). The position was valid for a short term, and could be awarded to the same person more than once. After 1518, there are no documents with signatures of the Abbot of the Monastery, but instead representatives of it, e.g. a judge, sacristan, prior, or simple hieromonks and elders.
In the 1394 Mt Athos Typikon (Book of rules) of Manuel Palaiologos, which was published in 1498 in order to regulate procedural issues relating to the general operation of the Monastery, also documents the situation on the peninsula at the end of the 15th century: The Karakallou Monastery held the seventh position, after Great Lavra, Vatopedio, Iviron, Chilandarios and Zeropotamos. From the same source, we learn that the Monastery was even required to give seven measures of wine and seven litres of olive oil to the Proto every year.
From the end of the 15th century and during the 16th, the Monastery seems to have maintained relationships with important personages of the era. In 1492, the leader Iakobos Malaspinas bought a precious artefact in Konstantinople, a 1290 calligraphic book with the four gospels, and repatriated it to the Monastery. In 1546, the Monastery was in communication with the Metropolite Makarios of Thessaloniki, who took up residence at the Vatopedio Monastery – where he lived as the monk Michael – shortly prior to 1527, after his resignation from the throne. During his time at the Monastery, he donated a large number of important manuscripts to the library, among which two manuscripts of the Παρακλητική (hymns for the Vespers) stand out, and are still considered to be significant today. At the same time, one of the most important ascetics of the 16th century, St Dionysios of Olympus, practised for some time at the border of the Monastery. According to information in his Life, he moved to a skete of the Monastery around 1520, constructed cells and the church of the Holy Trinity there, and took part in ascetic struggles. After his pilgrimage to Jerusalem, he returned to his skete, where he built the church of the Holy Fathers, before being elected as Abbot in the Filotheos Monastery.