The antimicrobial activity of the pygidial gland secretion released by adult individuals of the troglophilic ground beetle Laemostenus (Pristonychus) punctatus (Dejean, 1828), applying microdilution method with the aim to detect minimal inhibitory concentration, minimal bactericidal concentration and minimal fungicidal concentration, has been investigated. In addition, morphology of the pygidial glands is observed. We have tested 16 laboratory and clinical strains of human pathogens – eight bacterial both gram-positive and gram-negative species and eight fungal species. The pygidial secretion samples have showed antimicrobial properties against all strains of treated bacteria and fungi. Micrococcus flavus proved to be more resistant compared with other bacterial strains. More significant antimicrobial properties of the secretion are observed against Escherichia coli, which proved to be the most sensitive bacteria. Aspergillus fumigatus proved to be the most resistant, while Penicillium ochrochloron and Penicillium verrucosum var. cyclopium the most sensitive micromycetes. Commercial antibiotics Streptomycin and Ampicillin and antimycotics Ketoconazole and Bifonazole, applied as positive controls, showed higher antibacterial properties for all bacterial and fungal strains, except for P. ochrochloron, which proved to be more resistant on Ketoconazole compared with the pygidial gland secretion of L. (P.) punctatus. Apart from the role in ecological aspects, the antimicrobial properties of the tested secretion possibly might have medical significance in the future.