###### Abstract  
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 \(\pm\) 14.1 mL/g VS, containing 610 CH\({}_{4}\) mL/g VS, while in WH, the WH/CD ratio of 1:1 exhibited the highest biogas yield of 925 \(\pm\) 49.4 mL/g VS, with a methane content of 440 CH\({}_{4}\) mL/g VS. The co-digestion of the WH/FW ratio (1:1) showcased the highest biogas production of 1655 \(\pm\) 91.92 mL/g VS, accompanied by 890 \(\pm\) 70.7 CH\({}_{4}\) mL/g VS. This was followed by the 1:2 and 2:1 ratio, yielding 1400 \(\pm\) 56.5 and 1140 \(\pm\) 169.7 mL/g VS. of biogas and 775 and 585 CH\({}_{4}\) 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. a 2024 14 9880 17 October 2024 23 October digestion process, which collectively maximizes productivity and enhances the efficiency of organic fraction conversion [2; 3; 4]. ACoD also diminishes the impact of inhibitors and toxins, provides essential nutrients for microbial growth, shortens retention and lag times, and enhances both the loading rate and methane production [5]. Determining the optimal mixing ratio of substrates, as well as the inoculum-to-substrate ratio (ISR), is crucial for preventing the accumulation of volatile fatty acids (VFAs) and other inhibitors that can negatively impact the AcoD process and thus reduce the biogas potentiality [6]. Various studies have reported differing results regarding the influence of ISR on biogas and methane production during the AcoD of different feedstocks. Optimizing these parameters is essential for enhancing AcoD efficiency and maximizing biogas yields [7; 8; 9]. Anaerobic co-digestion of biological wastes serves as an effective substrate for biogas production, presenting a viable waste-to-energy technology. This process offers a straightforward approach to simultaneously address the growing demand for renewable energy sources and mitigate waste management challenges [10]. Biological waste, which can serve as a substrate for anaerobic co-digestion, includes a wide range of organic materials such as crop residues, agricultural residues [11], animal manure, sewage sludge, food waste [12], fish waste [13], etc. The Blue Revolution in India demonstrated the importance of the fisheries and aquaculture sector. The sector is considered as a sunrise sector and is poised to play a significant role in the Indian economy in the near future.