The main objective of this study was to optimize a culture media for low scale biomass production of Pleurotus spp. Future applications of this optimization will be implemented for ''in situ'' rice straw degradation, increase soil nutrients availability, and lower residue and rice culture management costs. Soil samples were taken from different points in six important rice production cities in Colombia. For carbon and nitrogen source selection a factorial 4 2 design was carried out. The Plackett-Burman design permitted to detect carbon, nitrogen and inducer effects on fungus growth (response variable for all designs). This optimization was carried out by a Box-Behnken design. Finally a re-optimization assay for glucose concentration was performed by means of a One Factor design. Only 4/33 (12 %) isolates showed and important laccase or manganese peroxidase activity compared to Pleurotus ostreatus (HPB/P3). We obtained an increased biomass production in Pleurotus spp. (T1.1.) with glucose, followed by rice husk. Rice straw was considered an inducing agent for lignin degradation. Glucose was a significant component with positive effects, whereas Tween 80 and pH had negative effects. On the contrary, rice husk, yeast extract and CaCl 2 were not significant components for increase the biomass production. Final media composition consisted of glucose 25 g L , and pH 4.88 ± 0.2. The Box-Behnken polynomial prediction resulted to be lower than the experimental validation of the model (6.59 vs. 6.91 Log 10 CFU ml -1 respectively).