Agro-ecosystems are intensively exploited environments which are both providers and consumers of ecosystem services. The improvement of both provisioning and regulating services in cultivated landscapes is crucial for the sustainable development of rural areas. Among the provisioning services offered, producing biogas from the anaerobic digestion of residual biomass is nowadays a promising option for decreasing greenhouse gas (GHG) emissions, while avoiding the land use conflicts related to the use of dedicated crops. Based on the available quantitative data at a regional level, provisioning and regulating services provided by the use of agri-food waste, livestock waste and agricultural residues were assessed for the case of Emilia Romagna region, the second biggest biogas producer in Italy. One provisioning service, i.e., bioenergy generation, and three regulating services were considered: (i) air quality improvement by the reduction of odors derived from direct use of waste, (ii) regulation of soil nutrients by reducing organic load and digestate spreading, and (iii) global climate regulation by saving GHG emissions. A potential further generation of 52.7 MW electric power was estimated at the regional level. Digestate spreading on fields may reduce odor impact by more than 90%, while containing a higher percentage of inorganic nitrogen, which is readily available to plants. The estimated GHG emission savings were equal to 2,862,533 Mg CO2eq/yr, mainly due to avoided landfilling for agri-waste and avoided replacing of mineral fertilizers for livestock waste and agricultural residues. The results suggest that bioenergy generation from lignocellulosic, livestock and agro-industrial residues may improve some regulating services in agro-ecosystems, while helping to reach renewable energy targets, thus contributing to overcoming the provisioning vs. regulating services paradigm in human-managed ecosystems.