This study was conducted to investigate the microorganisms associated with substrate used in the cultivation of mushroom ( Pleurotus ostreatus) i n a mushroom farm. The substrate which is a mixture of sawdust, rice bran, lime, and water was composted for 30 days and pasteurized for use in the cultivation. Samples of pasteurized substrates were randomly collected for the analyses using standard micro biological techniques. Microorganisms isolated and characterized and their frequency of isolation from the substrate include species of fungal genera Aspergillus (40.9%), Fusarium (22.7%), Mucor (5.6%) Penicillium (17.0%), Rhizopus (11.6%) and Trichoderma (2.3%). The species of bacteria genera isolated and frequency of isolation was Bacillus (36.67%) Clostridium (16.67%), Enterobacter (18.33%), Escherichia (15%), and Pseudomonas (13.33%). Aspergillus and Bacillus species had the highest frequency of occurre nce for fungi and bacteria respectively. Proper composting promotes the development of a number of saprophytic soil microorganisms that helps in the degradation of the substrate. The presence of cellulolytic fungi such as Aspergillus, Penicillium and Trichoderma are associated with the composting process and do accelerate composting for efficient recycling. Generally, the presence of fungi and bacteria in the pasteurized substrate is attributed to the ineffectiveness of the method adopted for pasteurization and the possession of spores that were heat tolerant by most of the isolates. The presence of E. coli which is an indicator of faecal contamination is attributed to the feacal contamination of the streams and river banks around the Sawmill where logs are retained before being sawn with the sawdust as a by - product. The presence of potential pathogens such as Bacillus , Clostridium , E. coli and Enterobacter can lead to bacterial disease of edible mushrooms and economic loss. The presence of these microorgani sms also has serious implications for human health.