Chromium (VI) is known to be harmful element that commonly found industrial waste, mining activities, and wastewater discharges from various industries. When released into the environment, Cr (VI) can contaminate soil, water, and air, posing a serious threat to living organisms. Aiming to Cr decontamination, this work was framed to isolate the fungal species having high Cr tolerance capacity and to exploit as bio-sorbent for the removal of Cr (VI) from aqueous solutions by biosorption. Among the fungal species isolated from the Cr (VI) contaminated soil sample, the filamentous fungus of Ophiocordycipitaceae family, Purpureocillium lilacinum was identified using molecular sequencing technique, showed maximum tolerance against Cr (VI) with a tolerance index of 1.19 ± 0.23. Further, Plackett Burman Design was applied to investigate for ascertaining the significance of different carbon and nitrogen sources on P. lilacinus growth, as well as the influence of environmental factors, such as pH, temperature, and Cr (VI) concentration. The results explicated that glucose was the most preferred carbon source for P. lilacinus, while yeast extract was the most preferred for nitrogen source. The optimum pH value and temperature were found to be 6.0 and 26 °C, respectively. In addition, P. lilacinus isolate was identified to survive in high concentrations of Cr (VI), indicating its potential for employing effective bioremediation of chromium-contaminated site.