The primary goal of this research is to isolate mycotoxin-producing fungus from the Nagavali River. Examining isolated fungi involved analyzing their mycelium growth on culture media and detailed microscopic inspection. We employed PCR analysis utilizing universal primers ITS1 and ITS4 to accurately identify the species. Furthermore, we sequenced the amplified ITS region and rigorously analyzed the sequences using NCBI-BLASTn and the ITS2 database. The analysis found a high 96.38% genetic similarity to the Aspergillus flavus strain, resulting in a 600-base pair fragment size. The sequence was given the accession number OR536222 in the NCBI GenBank database. Phylogenetic analysis was performed to ascertain the particular strain of A. flavus and its source. Remarkably, this analysis led to the identification of a single new strain gene, which represents a novel discovery in the field of fungal research. These results underscore the vital significance of molecular techniques in promptly and precisely identifying organisms. This research enhances our understanding of mycotoxin contamination in water, providing valuable insights to improve detection and prevention strategies. It accentuates the overarching importance of conserving our water resources and upholding ecological equilibrium, ultimately safeguarding the well-being of both humanity and the environment.