Most sources of biomass are located in rural areas, and use of this biomass for bioenergy may be in conflict with agricultural and conservation purposes. This study applied Life Cycle Inventory (LCI) and Geographical Information System (GIS) tools to estimate the Net Energy Gain (NEG) and Energy Return on Energy Invested (EROEI) for different biomass/bioenergy production activities in rural areas. The focus was on those activities that are least damaging or even socially, environmentally or economically beneficial. We considered bioenergy production from crop residues, manure, and grass in natural grasslands and surplus pasturelands. The feasibility and vulnerability of different sources was assessed within the context of existing policy constraints, and the potential contribution to the EU's bioenergy targets was evaluated. Taking the Overijssel province in the Netherlands as a case study, we showed that 66.01 PJ can be contributed from by-products, with an additional 3.34 TJ coming from more conventional pasturelands. The NEG from biogas can potentially take care of Overijssel's entire renewable energy target for the year 2030. When producing bioenergy from by-products, the EROEI is quite high (7–17), indicating that there is a big potential for by-products to provide energy without compromising the ecological or agricultural functions of the landscapes. However there are still many changes in the practices, technologies and policies associated with bioenergy production that have to be made to harvest this potential energy resource.