Usnea is one of the largest and most diverse genera of fruticose lichens with global distribution. Endolichenic fungi, which thrive within lichen thalli, have emerged as a promising source of bioactive compounds, with the ability to synthesise a variety of metabolites with biopharmaceutical potential. In this study, four isolates of endolichenic fungi isolated from Usnea spp. were identified using comprehensive multi-gene phylogenetic analyses. These isolates were evaluated for their anticancer, antifungal, and antibacterial properties, as well as for their ability to produce extracellular enzymes. Our findings revealed that the isolates represent four novel species, named as Amphisphaeria falcata, Kirschsteiniothelia tumidula, Neoroussoella annulata, and Veronaea brunneicolor. Our screening assay showed N. annulata and V. brunneicolor exhibited cytotoxic effects against the H460 human lung cancer cell line, with moderate inhibitory activity at a concentration of 100 μg/mL. The four fungal isolates exhibited distinct antifungal profiles against phytopathogens: Amphisphaeria falcata specifically inhibited Fusarium graminearum, while Veronaea brunneicolor showed broad-spectrum activity against Botrytis cinerea, F. graminearum, and Alternaria alternata. No antibacterial effects were detected in any isolates. These fungi exhibited a diverse array of extracellular enzyme activities, including amylase, protease, gelatinase, glucose oxidase, and cellulase. Collectively, these results underscore the considerable biotechnological potential of endolichenic fungi as sources of bioactive compounds with applications in drug discovery, agriculture, and environmental management. These findings also highlight the ecological importance of endolichenic fungi, suggesting that they may play multifaceted roles in lichen symbioses and their environments. Continued exploration of these fungi is essential for unlocking their full pharmacological and industrial potential.
