Abstract: Anaerobic digestion (AD) is a well-established process for converting organic waste into renewable bioenergy. However, optimizing methane potential remains a challenge due to feedstock variability and process limitations. This study presents a comparative and integrative evaluation of four agro-industrial biomasses Neem Deoiled Cake (NDC), Mango Kernel (MK), Waste Maize Flour (WMF), and Cow Dung (CD) by assessing their physicochemical profiles, calorific values, and essential trace metals. The objective is to investigate substrate synergies in co-digestion systems to maximize methane production. NDC displayed the highest calorific value (5219 kcal/kg) and favorable C:N ratio (12:1), while CD, despite its lower calorific value (3000 kcal/kg), provided superior buffering and microbial support due to high moisture and moderate nitrogen levels. WMF and MK offered high organic content but required co-substrates for process stability. Essential trace elements such as Fe, Zn, Cu, and Mn were detected in varying concentrations, influencing microbial metabolism and methane potential. The findings underscore the significance of balanced substrate combinations over individual calorific contributions and advocate for nutrient-based feedstock engineering for improved biomethane productivity.