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 MaguraCave, 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 а novel species. Heterotrophic bacterial isolates from MaguraCave were investigated for hydrolytic enzymes production, antimicrobial and hemolytic activity. Predominance of producers of protease (87%), followed by xanthan lyase (64%), lipase (40%), β-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 MaguraCave 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.