Lake Faro, in the North-Eastern corner of Sicily (Italy), shows the typical stratification of a meromictic tempered basin, with a clear identification of the mixolimnion and the monimolimnion, separated by an interfacial chemocline. In this study, an annual-scaled study on the space-time distribution of the microbial communities in water samples of Lake Faro was performed by both ARISA (Amplified Ribosomal Intergenic Spacer Analysis) and CARD-FISH (Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization) approaches. A correlation between microbial parameters and both environmental variables (i.e., temperature, pH, dissolved oxygen, redox potential, salinity, chlorophyll-a) and mixing conditions was highlighted, with an evident seasonal variability. The most significative differences were detected by ARISA between the mixolimnion and the monimolimnion, and between Spring and Autumn, by considering layer and season as a factor, respectively.