Experiment was carried out in mushroom research shade house of Olericulture Division, HRC (Horticulture Research Centre), Bangladesh Agricultural Research Institute, Joydebpur, Gazipur, during the period from July, 2015 to December, 2015 to evaluate the effect of pH levels of substrates and frequency of watering on the growth traits of oyster mushroom. Spawn of Pleurotus ostreatus (Jacquin ex Fr.) was used as test crop. The experiment consisted of two factors i.e., pH level of substrates [P0: Control (5.5), P1: 5.0, P2: 5.3, P3: 5.8, P4: 6.1 and P5: 6.4] and frequency of watering [W0: Control (No immersion in water and no further watering), W1: Im + 12h, W2: Im + 18h, W3: Im + 24h, W4: Im + 30h and W5: Im + 36h]. The experiment was laid out in Completely Randomized Design (CRD) with three replications. Results revealed that most of the parameters showed the significant response due to different pH levels of substrate and watering frequency. For pH level of substrate, the treatment P0 (5.5) exhibited the better performance on the growth characters of mushroom. In case of watering frequency, the treatment W1(Im + 12h) showed higher number of fruiting bodies and higher number of harvest of mushroom. Statistically, the highest number of fruiting bodies was obtained from the combined treatment P0× W1 i.e., pH of substrate 5.5 in combination with watering frequency Im + 12 h. So, this combination may be used for higher yield of mushroom.