Biocapacity of bacteria inhabiting karstic caves to produce valuable biologically active compounds is still slightly investigated. A total of 46 culturable heterotrophic bacteria were isolated under aerobic conditions from the Gallery with pre-historical drawings in Magura Cave, Bulgaria. Phylogenetic analysis revealed that most of bacterial isolates affiliated with Proteobacteria (63%), followed by Actinobacteria (10.9%), Bacteroidetes (10.9%), and Firmicutes (6.5%). A strong domination of Gram-negative bacteria (total 81%) belonging to nine genera: Serratia, Pseudomonas, Enterobacter, Sphingobacterium, Stenotrophomonas, Commamonas, Acinetobacter, Obesumbacterium, and Myroides, was observed. Gram-positive isolates were represented by the genera Bacillus, Arthrobacter, and Micrococcus. One isolate showed a significant phylogenetic distance to the closest neighbor and could represent. novel species. Heterotrophic bacterial isolates from Magura Cave were investigated for hydrolytic enzymes production, antimicrobial and hemolytic activity. Predominance of producers of protease (87%), followed by xanthan lyase (64%), lipase (40%), beta-glycosidase (40%), and phytase (21%) was observed. Over 75% of the isolates demonstrated antimicrobial and hemolytic activity. The results suggest that heterotrophic bacteria isolated from Magura Cave could be a valuable source of industrially relevant psychrotolerant enzymes and bioactive metabolites. This study is a first report on the taxonomic composition and biological activity of culturable bacteria inhabiting a cave in Bulgaria.