The Monastery of Hosios Loukas is situated at an altitude of 430 m (38°23′43.12′′N, 22°44′48.22′′E) in the western foothills of Mount Helikon, near the village of Steiri, Boeotia, Greece. It is one of the most important monuments of Middle Byzantine art and architecture and has been recognized by UNESCO as a World Heritage Site. Information varies concerning the construction date of the magnificent church to house the Hosios Loukas relics. Moreover, little is known about the time when the rest of the monastic complex was built. This paper aims to shed light on these chronological questions. For this purpose, 15 wood samples, originating from the outermost rings of the wooden timber preserved from the initial scaffolding of the church and its four supporting buttresses and another two from the wooden gate of the monastery, were radiocarbon (14C) dated using the conventional gas-counting technique. Our results show that the church was built in the beginning of the 11th century. The four buttresses were built in at least two phases, during the 15th through 19th centuries, and the monastery gate may be also attributed to the 19th century.