The aim of this study was the isolation and molecular characterization of fungi from untreated refinery effluent by using multiple conserved genes. The Fungi isolated were characterized based on PCR amplification and genomic sequencing of the internal transcribed spacer region (ITS), partial β‐tubulin (BenA), calmodulin (CaM), and RNA polymerase second large subunit (RPB2) genes, along with morphological characterization. The obtained sequences were subjected to BLAST analysis and the corresponding fungal isolates were assigned species names after comparison with representative sequences available in GenBank. Fifteen (15) Fungi species belonging to four genera of Aspergillus, Penicillium, Fusarium, and Trichoderma with Aspergillus as the predominant genus were identified. Therefore these genes should be used as molecular markers for species level identification of fungi (especially Aspergillus and Penicillium as proven in this study.