In this article, five variables including type of substrates, sizes of substrates, mass ratio of spawn to substrates (SP/SS), temperature and pretreatment of substrates were used to model mycelium growth in Pleurotus sp. (oyster mushroom) cultivation by using agricultural wastes via two level factorial analysis. Two different substrates which were empty fruit bunch (EFB) and sugarcane bagasse (SB) were used. Analysis of Variance (ANOVA) for both mycelium extension rate (M) and nitrogen concentration in mycelium (N) showed that the confidence level was greater than 95% while p-value of both models were less than 0.05 which is significant. The coefficient of determination (R2) for both M and N were 0.8829 and 0.9819 respectively. From the experiment, the best condition to achieve maximum M (0.8 cm/day) and N (656 mg/L) was by using substrate B, 2.5 cm size of substrate, 1:14 for SP/SS, incubated at ambient temperature and application of steam treatment. The data showed that EFB can be used to replace sawdust as a media for the oyster mushroom cultivation. Data analysis was performed using Design Expert version 7.0.