<p>Biological pretreatment of rice straw using <i>Pleurotus ostreatus</i> was evaluated based on the physicochemical changes and degradation kinetics of lignocellulosic components. Inoculum concentrations of 5%, 10%, and 15%, along with incubation times of 7, 14, 21, 28, and 35 days, were employed to assess the degradation of lignin, cellulose, and hemicellulose in rice straw. The maximum degradation rates for lignin, cellulose, and hemicellulose were accomplished at an inoculum concentration of 15% and an incubation time of 35 days, resulting in degradation percentages of 37.50%, 19.78%, and 14.75%, respectively. The first-order kinetic model has successfully described the kinetics of lignocellulose degradation, demonstrating a high coefficient of determination (R²). The degradation rate constant (k) for lignin increased by 13.49% when the inoculum concentration surged from 5 to 10%. Further increasing the inoculum concentration from 10 to 15% resulted in a 6.99% increase in the k value. An effective and efficient fungal pretreatment was accomplished at an inoculum concentration of 15% and an incubation time of 21 days, yielding lignin removal, cellulose retention, and hemicellulose retention rates of 31.80%, 93.25%, and 88.35%, respectively. The success of the pretreatment was corroborated through chemical and structural analysis of rice straw, employing Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and Brunauer-Emmett-Teller (BET). These findings indicate that <i>P. ostreatus</i> can effectively and efficiently degrade lignin in a more environmentally friendly pretreatment process.</p>