Lacustrine sediments are widely used to investigate the impact of climatic change on biogeochemical cycling. In these sediments, subsurface microbial communities are major actors of this cycling but can also affect the sedimentary record and overprint the original paleoenvironmental signal. We therefore investigated the subsurface microbial communities of the oldest lake in Europe, Lake Ohrid (North Macedonia, Albania), to assess the potential connection between microbial diversity and past environmental change using 16S rRNA gene sequences. Along the upper ca. 200 m of the DEEP site sediment record spanning ca. 515 thousand years (ka), our results show that Atribacteria, Bathyarchaeia and Gammaproteobacteria structured the community independently from each other. Except for the latter, these taxa are common in deep lacustrine and marine sediments due to their metabolic versatility adapted to low energy environments. Gammaproteobacteria were often co-occurring with cyanobacterial sequences or soil-related OTUs suggesting preservation of ancient DNA from the water column or catchment back to at least 340 ka, particularly in dry glacial intervals. We found significant environmental parameters influencing the overall microbial community distribution, but no strong relationship with given phylotypes and paleoclimatic signals or sediment age. Our results support a weak recording of early diagenetic processes and their actors by bulk prokaryotic sedimentary DNA in Lake Ohrid, replaced by specialized low-energy clades of the deep biosphere and a marked imprint of erosional processes on the subsurface DNA pool of Lake Ohrid.