The emergence of antibiotic resistance is a global health crisis, thus the search for novel antimicrobial compounds has become a continuous necessity. Underexplored and extreme environments, such as cave ecosystems, have been identified as a promising potential source for the discovery of novel microorganisms with novel antimicrobial compounds (AMC). This study presents the first cave microbiological investigation in Sri Lanka, with a special preference for bioprospecting of novel AMC. The cave sediment characterization demonstrated the presence of close to strong acidic conditions (pH 3.1 – 3.3) and thus indicates the possibility of isolating acidophilic microorganisms. Eight cave wall/ceiling fungal strains were isolated from Sthreepura Cave - Kuruwita and identified using both morphological and ribosomal Internal Transcribed Spacer (ITS) region sequence analysis. Interestingly, four fungal isolates (Penicillium panissanguineum, P. cremeogriseum, Aspergillus bertholletius and Trichoderma yunnanense) were found to be the first records in Sri Lanka. Of these eight isolates, three showed antimicrobial activity (AMAs) against at least one of the five tested human pathogens in preliminary screening, while A. fumigatus (SKW 404) strain showed the highest AMA against Staphylococcus aureus (ATCC 11778) assessed by agar culture plug method on Muller Hinton Agar (MHA). Crude Ethyl Acetate (EtOAc) fraction of both mycelial and Potato Dextrose Broth (PDB) extracts of A. fumigatus demonstrated similar bioactive metabolic profiles with four corresponding chemical fractions [Rf = 0.47, 0.56, 0.65, 0.82; EtOAc: Hexane (4:1, v/v)] in TLC: agar overlay bioassay. The present study indicates that there is potential for discovering novel Sri Lankan deep cave microorganisms and bioprospecting of their novel bioactive compounds. Hence, further island-wide in-depth cave microbiological investigations are required for a better understanding of the Sri Lankan cave microbiology
