Pest infestations present a significant challenge to agricultural productivity, and the excessive, indiscriminate utilization of chemical insecticides has led to issues like pest resistance, the emergence of new pest species, and serious environmental concerns. Consequently, adopting biopesticides is vital for sustainable pest management strategies. Entomopathogenic fungi (EPF) are efficient biological control agents that regulate pests through direct contact and penetration in the host’s cuticle, releasing toxins and proliferating by utilizing nutrients in the haemocoel while evading the host’s immune defences. Researchers have aimed to create effective fungal biocontrol formulations for managing plant diseases and pest infestation. One such formulation, “Biomix” was developed by VNMKV Parbhani. This microbial consortium contains beneficial microorganisms, including EPF and decomposer fungi such as Beauveria bassiana, Lecanicillium lecanii, Metarhizium anisopliae, and Aspergillus niger. These microbes collectively improve soil health, crop productivity through nutrient cycling, pathogen suppression and pest control. Given the significant role of “Biomix” it is essential to characterize these fungal isolates for integrated pest management. In this study, fungal isolates underwent morphological characterization, documenting growth patterns, colony color, texture, sporulation time, and hyphal structure. Molecular characterization involved extracting genomic DNA from mycelial mats, followed by PCR amplification of ITS rDNA region using ITS-1/ITS-4 primers. Based on the query coverage (QC), E-value, as well as sequence similarity, Beauveria bassiana isolate VNMKV 1 demonstrated the highest similarity (99.65%) to Beauveria bassiana strain 23D112, the Lecanicillium lecanii isolate VNMKV 1 showed 99.65% similarity to Lecanicillium lecanii isolate Vl-5, Aspergillus niger isolate VNMKV 1 exhibited the highest similarity (99.33%) with Aspergillus sp. isolate AUMS56 and Metarhizium anisopliae isolate VNMKV 1 displayed the highest similarity (99.82%) with Metarhizium anisopliae strain CN094G8.