The south-dipping Gyrtoni Fault defines the northeastern boundary of the Middle-Late Quaternary Tyrnavos Basin, Central Greece. The recognition and recent tectonic activity of the fault were previously based on mapping, remote sensing analyses and electrical resistivity tomography studies. To understand the Holocene seismotectonic behavior of the Gyrtoni Fault we excavated two paleoseismological trenches. To estimate the timing of past earthquakes using luminescence dating, we obtained twenty five fluvial-colluvial sediment and pottery samples from both the upthrown and the downthrown fault blocks. We applied the Optically Stimulated Luminescence (OSL) dating to coarse grain quartz using the single-aliquot regenerative-dose (SAR) protocol. Our investigations of luminescence characteristics using various tests confirmed the suitability of the material for OSL dating. We found that the estimated OSL ages were internally consistent and agreed well with the available stratigraphical data, archaeological evidence and radiocarbon dates. The performed paleoseismological analysis emphasized the occurrence of three surface faulting events in a time span between 1.42 ± 0.06 ka and 5.59 ± 0.13 ka. Also, we recognized an earlier faulting event (fourth) has been also recognized to be older than 5.59 ± 0.13 ka. The mean throw per event value of 0.50–0.60 m could correspond to a ca. Mw 6.5 earthquake. An average fault slip rate of 0.41 ± 0.01 mm/a and an average recurrence time of 1.39 ± 0.14 ka were also estimated. Our results suggest that the elapsed time from the most recent event (minimum age 1.42 ± 0.06 ka) is comparable with the mean return period.