Benzo[a]pyrene (BaP) is a highly toxic organic pollutant widely distributed in terrestrial and aquatic environments and is often referred as a xenobiotic compound. The present investigation was carried out to determine the physical factors affecting the biodegradation of BaP by the PO-3 isolate of Pleurotus ostreatus. When tested for the effect of media pH on the extent of BaP degradation, pH 6.0 facilitated both highest degradation (63.1%) and fungal biomass accumulation. Mesophilic range of temperature in general supported good fungal growth and subsequent degradation of the toxin. However, the highest degradation (64.2%) was observed when the inoculated media was incubated at 30°C. Subjecting the mineral salt broth to different agitation speeds showed different levels of BaP removal, wherein 180 rpm resulted in the highest level of BaP degradation (66.3%). Eventually, increasing the agitation speed beyond this decreased both the biomass and the extent of BaP degradation. Thus, the biodegradative potential of Pleurotus ostreatus PO-3 is strongly affected by these physical factors. A better understanding of the other parameters may result in effective decontamination of the polluted sites using Pleurotus ostreatus PO-3 isolate.