The presence of mycobacteria was studied in Bull Rock Cave (“Byci skala”) and the water catch - ment area of Jedovnice Brook (“Jedovnicky potok”) using direct microscopy after Ziehl-Neelsen (ZN) staining, culture examination and molecular techniques. Mycobacteria were detected in 47.1% of a total of 68 samples. The mycobacterial genes hsp65 and dnaA were detected and sequenced in 37 (74.0%) out of the 50 cave environmental samples and in 10 (55.6%) out of the 18 samples of water catchment sediments. Nine species of slowly growing mycobacteria ( M. terrae , M. arupense , M. gordonae , M. lentiflavum , M. parascrofulaceum , M. parmense , M. sas - katchewanense , M. simiae and M. xenopi ) and two subsp. ( M. avium subsp. avium and M. avium subsp. hominis - suis ) were detected. Fourteen species of rapidly growing mycobacteria ( M. chelonae , M. chubuense , M. poriferae , M. flavescens , M. fortuitum , M. porcinum , M. rhodesiae , M. gilvum , M. goodii , M. peregrinum , M. mageritense , M. vanbaalenii , M. gadium and M. insubricum ) were detected. The highest mycobacterial presence was docu- mented by ZN staining and/or culture examinations in earthworm castings and bat guano (73.3% positivity out of the 15 samples) in the cave environment and in the water sediments collected under the outflow from the wastewater treatment plants (77.8% positivity out of nine samples). The highest total organic carbon (TOC) was detected in wooden material and earthworm castings with pH values between 5.0 and 7.7 in the cave environment and in water sediments collected under the outflow from the wastewater treatment plants with pH between 5.8 and 7.0. It could be concluded that the karst cave environment with its running surface water contaminated with different microorganisms or chemical substances creates favourable conditions not only for animals (especially earthworms) but also for mycobacteria. This fact is also demonstrated by the presence of these mycobacteria in the cave environment mainly in earthworm castings and bat guano.