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.