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Abandoned and flooded ore mines are examples of hostile environments (cold, dark, oligotrophic, trace metal) with a potential vast diversity of microbial communities rarely characterized. This study aimed to understand the effects of depth, the source of water (surface or groundwater), and abiotic factors on the communities present in the old Forsyth iron mine in Quebec (Canada). Water and biofilm samples from the mine were sampled by a team of technical divers who followed a depth gradient (0 to 183 m deep) to study the planktonic and sessile communities' diversity and structure. We used 16S/18S rRNA amplicon to characterize the taxonomic diversity of Bacteria, Archaea, and Eukaryotes. Our results show that depth was not a significant factor explaining the difference in community composition observed, but lifestyle (planktonic/sessile) was. We discovered a vast diversity of microbial taxa, with taxa involved in carbon- and sulfur-cycling. Sessile communities seem to be centered on C1-cycling with fungi and heterotrophs likely adapted to heavy-metal stress. Planktonic communities were dominated by ultra-small archaeal and bacterial taxa, highlighting harsh conditions in the mine waters. Microbial source tracking indicated sources of communities from surface to deeper layers and vice versa, suggesting the dispersion of organisms in the mine, although water connectivity remains unknown. [ABSTRACT FROM AUTHOR]