Different inoculum to slaughterhouse waste (SHW) ratios (Ino/SHW) influences the digester performance, substrate utilization, and methane yield through microbial shift and their metabolic syntrophy. Acetoclastic Methanosarcina (68–87%) was dominant in the exponential phase, overpowering the initial abundance of Methanosaeta (86% of methanogens) in the SHW digesters. Positive interactions among acetogenic and acetate-oxidizing species of Clostridium (11%) with Methanosarcina (84% of methanogens) improved the methanogenic activity (292 mL g⁻¹ VS_initial d⁻¹) and final VS utilization (90%) at the highest Ino/SHW loading. In contrast, significant improvement of methane yield (152% higher than the control) at the lowest Ino/SHW loading was attributed to strong syntrophy among Methanosaeta (24% of methanogens) and its exoelectrogenic partners, Bythopirellula (0.52%) and Mariniphaga (0.08%) and the acetogenic Cloacimonas (0.16%) and Longilinea (0.32%). These syntrophic interactions among the core microbiota induced major metabolic activities, including butanoate, glycine, serine and threonine, methane, propanoate, and pyruvate metabolism, and quorum sensing.