Fungi represent a vast and largely unexplored source of biologically active metabolites. This study aimed to isolate fungi from ecologically distinct regions of the Republic of Azerbaijan and evaluate their antioxidant and antimicrobial potential. During 2022–2025, 105 pure cultures were obtained from soil, water, and plant samples collected across the Greater Caucasus, Lesser Caucasus, Kura–Araz lowland, and the southern region (Lankaran–Astara), including 79 micromycetes and 26 macromycetes. Initial screening of exogenous metabolites revealed variable activity profiles: among micromycetes, antioxidant activity was classified as weak (32 strains), medium (27), and strong (5), while antimicrobial activity was weak (26), medium (37), and strong (4). Comparable variability was observed in macromycetes. Four highly active strains were selected for detailed study. Based on classical and ITS rDNA analysis, the most promising micromycetes were identified as Trichoderma citrinoviride AEF-2024 and T. harzianum AEF-2024 (99.83% and 99.66% sequence similarity, respectively). Optimization experiments demonstrated that liquid Čapek-Doks medium yielded maximum biomass (12.5 and 10.7 g/L for T. citrinoviride and T. harzianum, respectively). Under optimized conditions, culture solution (CS) exhibited pronounced antimicrobial activity (34–35 mm inhibition zones), whereas biomass extracts (BM) showed higher antioxidant capacity (29.6–31.2% DPPH radical scavenging). The findings highlight significant strain-level variability and confirm that fungal isolates from diverse Azerbaijani ecosystems constitute promising sources of bioactive metabolites with potential biotechnological and pharmaceutical applications.
