This study examines the impact of different inoculum-to-substrate ratios on biohydrogen production using various waste materials. The research aims to optimize biohydrogen yield by analyzing different combinations of agricultural, industrial, and animal wastes as substrates in anaerobic fermentation. A lab-scale study was conducted using 250 mL serum bottles, with the experiment running for 30 days to assess biohydrogen production. The substrates used in the study included rice husk, sugarcane waste, cow dung, and anaerobic sludge. The effect of different inoculum-to-substrate ratios on biohydrogen yield was also examined by varying inoculum concentrations (1, 1.5, 2, 2.5, and 3% by volume) in the fermentation process. The maximum cumulative biohydrogen yield was achieved from specific mixed ratios of substrates. The highest yield, 12.6 mL, was observed in the rice husk and anaerobic sludge combination. This was followed by 9.3 mL of biohydrogen from sugarcane waste and anaerobic sludge and 8.1 mL from sugarcane waste and cow dung. These results indicate that substrate composition and inoculum concentration significantly influence the biohydrogen production potential. This study demonstrates the effective use of different waste materials for sustainable biohydrogen production, highlighting the potential for scaling up the process for cost-effective, pilot-scale applications. The findings contribute to developing waste-to-energy strategies, focusing on using agricultural, industrial, and animal waste in biohydrogen production, which can provide a sustainable energy alternative to traditional energy sources. The research also suggests that optimizing inoculum-to-substrate ratios can enhance the overall efficiency of the biohydrogen production process.