Abstract The Masalli Istisu thermal springs in southern Azerbaijan represent a unique geothermal ecosystem characterized by mineral-rich waters with temperatures ranging from 45–60°C. Such conditions provide an ideal environment for thermophilic and thermotolerant microorganisms with ecological and biotechnological significance. This study investigated the microbial diversity of Masalli Istisu, with a focus on Bacillus spp. and filamentous fungi, during spring (April–May 2025) and autumn (September–October 2025). Water and sediment samples were collected from four representative sites, and microorganisms were isolated on Maltose Peptone Agar (MPA) and Potato Dextrose Agar (PDA). Morphological, Gram staining, and colony enumeration analyses were conducted to assess microbial composition and seasonal variation. Results demonstrated that Bacillus spp. were the dominant microbial population, consistently detected at high colony-forming unit (CFU) counts (1.1 × 10⁵–1.4 × 10⁵ CFU/ml) across both seasons. Gram staining confirmed their Gram-positive rod-shaped morphology, occurring singly or in short chains. Filamentous fungi, including Alternaria, Aspergillus, and Penicillium species, were isolated at lower abundances, with slightly higher counts in spring compared to autumn. Seasonal fluctuations in fungal abundance are likely influenced by environmental factors such as temperature shifts, nutrient availability, and hydrological conditions, whereas Bacillus populations remained relatively stable. The findings highlight the ecological resilience of Bacillus spp. in thermophilic environments and the complementary role of fungi in ecosystem stability. Both groups possess biotechnological potential: Bacillus strains for thermostable enzyme production, antimicrobial compounds, and bioremediation, and fungi for bioactive metabolites and industrial enzyme applications. Overall, Masalli Istisu represents a promising geothermal habitat for future molecular and metagenomic exploration of novel thermophilic microorganisms.