This study is the first broad investigation of antimicrobial activity of different development stages of Coprinus cinereus from Tanzania. The indigenous edible wild C. cinereus mushroom was successfully grown in tropical conditions on dried grasses supplemented with different amounts of cow dung manure. Ethyl acetate crude extracts were prepared from different developmental stages of the mushroom, and were screened for antifungal and antibacterial activities using agar well method. Different extracts from black caps, post capping stage and black stem exhibited activity against Escherichia coli, Candida albicans and Aspergillus niger none of the gram positive bacteria was inhibited to grow. Differences in the substrate composition presented differences in the bioactivity of the mushroom extracts with 2: 3 ratio of cow-dung: grasses producing the highest activity. The results obtained clearly indicated that Tanzanian C. cinereus extracts contains bioactive components and are potential sources of antimicrobial compounds that could be used for development of new agents for the treatment and prevention of diseases.