The effect of pure and mixed substrates (medium) on growth and yield of oyster mushroom (Pleurotus ostreatus) was investigated. Seven different substrates namely cotton waste only (CWO), Sawdust only (SDO), Banana leaves only (BLO), Cotton waste + sawdust (CWSD), Cotton waste + Banana leaves (CWBL), Sawdust + Banana leaves (SDBL), and Cotton waste + Sawdust + Banana leaves (CWSDBL) were used. The substrates were pasteurized with hot water (90°C for 4 h) before spawns of oyster mushroom were inoculated to them. After inoculation, the substrates were kept in a controlled environment until fruiting took place. The CWO substrate gave the highest number (35) of fruiting body and highest yield (928.0 g). This was followed closely by the harvest from CWSDBL substrate. There was no fruiting recorded (0.0 g) in BLO substrate for oyster mushroom (Pleurotus ostreatus). The major conclusion from the research was that cotton waste, sawdust, are better substrates for mushroom cultivation whether in mixed or pure forms, meanwhile banana leaves are therefore not suitable for oyster mushroom (Pleurotus ostreatus) cultivation unless used in combination with either of the other two substrates.