The present study has been carried out to isolate mycoflora associated with Pleurotus ostreatus (Oyster mushroom) substrate during culture in the spawn packet. A total of 50 spawn packets with colonized substrate of Pleurotus ostreatus were collected randomly at different growth stages from Mushroom Culture Center, Savar, Dhaka. Ten mycoflora including Aspergillus flavus, A. fumigatus, A. niger, A. nidulans, A. terreus, Penicillium citrinum, P. thiersii, Penicillium. sp., Rhizopus stolonifer and Trichoderma harzianum were isolated and identified as associated with Pleurotus ostreatus (Oyster mushroom) substrate. Prevalence of all the isolated fungi gradually increased from mother spawn stage to 3rd flush stage. The growth of R. stolonifer was the quickest followed by T. harzianum. A laboratory trial has been conducted with Formalin, Bavistin and combination of Formalin and Bavistin at three different concentrations against identified associated mycoflora of Oyster mushroom substrates. The combination of Formalin and Bavistin at the highest concentration (500+75 ppm) was found to be the best in inhibiting the radial colony growth of all the identified fungi.