Three consortia were developed out of the ligninolytic fungal isolates for pretreatment of rice straw. The fungal consortium of Pleurotus ostreatus and Phanerochaete chrysosporium, produced maximum laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP) activities of 2.40 nkat/gds, 37.92 nkat/gds, and 62.50 nkat/gds, respectively. Response surface methodology (RSM) based on central composite design (CCD) was used to optimize pretreatment of rice straw under solid state conditions. With a desirability value of 0.862, the optimum conditions (moisture, 121.1%; temperature, 31.3°C; and log spore count, 8.0 spores/mL) resulted in 43.0%, 12.7% and 7.0% cellulose, hemicellulose, and lignin, respectively, from an initial respective values of 39.5%, 23.3%, and 11.5%. Optimization under shake flask conditions using 2.5 g rice straw, 8.0 mL concentrated ligninolytic enzyme extract, 10 mM Ca2+ ions, 45°C incubation temperature in an incubation period of 72 h resulted in 56.4%, 12.2%, and 2.2%, cellulose, hemicellulose, and lignin, respectively. © 2018 American Institute of Chemical Engineers Environ Prog, 38: 635–646, 2019