This study evaluates the performance of different agricultural by-products to identify the potential effect of independent variables, using as the dependent variable the biogas production. A Box–Behnken experimental design was carried out in a pilot-scale plant of four stirred stainless-steel digesters under mesophilic semi-continuous digestion. The results obtained support the creation of a technical framework to scale up the process and further evaluation of the potential environmental impacts through life cycle assessment (LCA) methodology. A stable behaviour was achieved in 12 of the 13 experiments proposed. The highest value of daily biogas production was 2200.15 mL day⁻¹ with a stabilization time of 14 days, an organic loading rate of 4 g VS feed daily, low C/N ratio and a 1:1 relation of nitrogen providers. The concentrations of CH₄ remained stable after the production stabilization and an average biogas composition of 60.6% CH₄, 40.1% CO₂ and 0.3% O₂ was obtained for the conditions mentioned above. Therefore, the real scale plant was estimated to manage 2.67 tonnes of residual biomass per day, generating 369.69 kWh day⁻¹ of electricity. The LCA analysis confirms that the co-digestion process evaluated is a feasible and environmentally sustainable option for the diversification of the Colombian energy matrix and the development of the agro-industrial sector.