The current investigation explores biogas production from water hyacinth (WH) and fish waste (FW) with cow dung (CD) as an inoculum source in two scenarios. In the first scenario, the optimization of mono-digestion was performed where the effect of WH/FW (substrates) with CD (inoculum) in varied ratios of 1:1, 1:2, 2:1, and 3:1 was observed to enhance the biogas production. In the second scenario, the optimization of co-digestion using both FW and WH as substrates in different ratios (1:1, 1:2, and 2:1) with a fixed amount of inoculum was studied. The experiments were conducted in 500 mL digesters in duplicate under mesophilic conditions. Under mono-digestion conditions for FW, the digester operating with FW/CD in a 1:2 ratio demonstrated the highest biogas yield of 970 ± 14.1 mL/g VS, containing 610 CH4 mL/g VS, while in WH, the WH/CD ratio of 1:1 exhibited the highest biogas yield of 925 ± 49.4 mL/g VS, with a methane content of 440 CH4 mL/g VS. The co-digestion of the WH/FW ratio (1:1) showcased the highest biogas production of 1655 ± 91.92 mL/g VS, accompanied by 890 ± 70.7 CH4 mL/g VS. This was followed by the 1:2 and 2:1 ratio, yielding 1400 ± 56.5 and 1140 ± 169.7 mL/g VS. of biogas and 775 and 585 CH4 mL/g VS, respectively. The CD and WH mixture at a 1:1 ratio demonstrated the most significant decrease in chemical oxygen demand (COD), reaching 91.68%. COD reductions over 80% in all combinations were observed in all instances. Anaerobic digestion (AD) simulations were validated using the Gompertz model, with high correlation coefficient values (R-squared) above 0.99 for all of the studied ratios, depicting a significant correlation between experimental data and model predictions. The propionic to acetic acid ratio did not cross the threshold level, indicating no inhibition of methane production. ANOVA analysis of biogas production between the co-digestion and mono-digestion of substrates showed non-significant results (p > 0.310 and p > 0.824, respectively), while overall digestion was significant (p < 0.024), indicating efficiency variations among substrates. Paired sample t-tests revealed substantial differences between co-digestion ratios, which were also significant.