Anaerobic digestion (AD) of agricultural wastes is a promising approach for energy recovery and crop residue management. However, its recalcitrant chemical structure hinders microbial hydrolysis and reduces biomethane production under AD. Biochar supplementation has been proven to promote the digestibility and biomethanation of lignocellulosic substrates. Therefore, this study investigated the influence of different pyrolysis temperatures (450 °C, 550 °C, and 650 °C) on the physicochemical properties of biochar. Furthermore, the impact of ruminal content biochar supplementation (1 %, 2 %, and 3 %) on the AD of rice straw with rumen fluid as inoculum has been investigated. The ruminal content biochar (RUCB) supplemented reactors showed an increment in biomethane yield and the highest cumulative biomethane yield 243.11 mL/g volatile solids (VS)) was recorded at 2 % RUCB supplementation, followed by 227.12 mL/g VS at 1 % RUCB supplementation and 162.86 mL/g VS at 3 % RUCB supplementation (P > 0.05). Compared to the control reactors (128.68 mL/g VS), RUCB supplemented reactors exhibited 1.88-fold, 1.76-fold, and 1.26-fold increments in biomethane yield due to pH stabilization and facilitation of microbial biofilm formation on the biochar. The correlation analysis showed that biomethane production is positively correlated with VS reduction (R² = 0.9852). This study proposed a potential strategy to utilize ruminal content waste as a feedstock for biochar production and its application in AD for accelerating the biomethanation of rice straw.