Agro-industrial transformation chains generate huge amounts of by-products which can be used as biomass for anaerobic digestion (AD) process, by avoiding their disposal in landfill and therefore environmental issues. This study has investigated a biomass mix to increase biogas production optimizing AD process parameters with the aim of enhancing AD performance in terms of produced biogas quality. In detail, both Biochemical Methane Potential (BMP) and AD approaches were adopted for investigating the effect of mixing seven Mediterranean feedstocks (i.e., olive pomace, citrus pulp, poultry and cattle manure, whey, tomato peels, and cereal straw) on methane production for bioenergy generation. The BMP test demonstrated that the tested mix had potential to be used for biogas production with 60.9% of methane content in the produced biogas. In detail, the specific production was equal to 343.1 Nm3CH4/tVS, confirmed by the results obtained during the AD test, about 333 Nm3CH4/tVS. As result, the tested mix demonstrated good net energy outputs and provide a flexible and useful solution to generate bioenergy from by-products. These findings confirm the possibility of using these by-products as energy sources and initiating virtuous bioeconomy processes for the sustainable utilization of renewable natural resources, transforming waste-to-resources.