The Tanzanian edible mushroom species Coprinus cinereus was grown on sisal waste substrates supplemented with chicken manure with the aim to evaluate the effects of the chicken manure supplement on the antimicrobial activity of the mushroom’s extracts. Crude ethyl acetate extracts were prepared from the mushroom’s fruiting bodies harvested at pre-capping, capping and post capping stages, and the extracts were tested for antimicrobial activity, using the agar well method. The antimicrobial activity was observed only in capping and post capping stages of the mushrooms and the activity generally increased with increased percentage of manure supplementation. These findings show that Tanzanian edible C. cinereus mushroom contains antimicrobial compounds and chicken manure could be used in the cultivation of the mushroom to increase the production of active secondary metabolites, which could be used as lead compounds for discovery of new and more effective drugs against microbial infections.