Prokaryotes from different phylogenetic groups were studied in surface microlayer (SM, up to 100 μm) and subsurface water (SW – 20 cm) in a eutrophic lake over three months (July, August, and October). The abundance of prokaryotes was determined by epifluorescence microscopy after DAPI staining, and phylogenetic diversity was determined by fluorescence in situ hybridization (FISH) with group-specific, fluorescently labeled oligonucleotide probes. In SW bacteria made up most of the entire community of DAPI-stained microorganisms (54-69%) and in SM bacteria made up only 33-44% of DAPI-stained microorganisms. Archaea corresponded to a small fraction of both bacterioneuston and bacterioplankton. The counts of Archaea and bacteria were significantly higher in SW than in SM. Among all proteobacteria included in the research, γ-proteobacteria represented the most abundant fraction: 42-72% in SM and 39-61% in SW. Statistical analysis revealed that the abundance of γ-proteobacteria is positively correlated with temperature and with dissolved oxygen. β-proteobacteria were the least abundant fraction.