Biochar has attracted increasing attention as an additive for enhancing the performance of anaerobic digestion (AD), but the effect of biochar on microbial regulatory mechanisms in enhancing AD performance is unclear. To investigate how biochar modulates the process of AD, different inoculum sources including cellulose–peptone–swine inoculum (CPI) and swine manure inoculum (SMI) were designed to determine the effect of biochar on the performance and microbial communities of anaerobic digestion of the feedstock concentration from 1 to 6%. The results showed that the methane yields of CPI seeds were higher 20.3–38.7% than those of SMI seeds without a biochar addition, whereas the biochar addition reduced 5.3 and 23.1% of the corresponding methane yield of CPI and SMI, respectively. The biochar enhances the accumulation of volatile fatty acids (VFAs) and weakens the potential ammonia inhibition by adsorption, and it can improve the degradation rate of organic content of soluble COD for different inoculum sources. Microbial community analyses showed that the biochar addition could facilitate the growth of Bacteroidetes and Clostridiales, and it enriched the relative abundance of hydrogenotrophic methanogens Methanobrevibacter and Methanobacterium. Overall, although the modulation of biochar possessed different effects on the anaerobic digestion performance, it contributed to the stability and degradation efficiency of the digestion system. The recycling implication of biochar is critical to realizing a low-carbon and renewable treatment system for organic wastes.