The production of bioenergy is dependent on the supply of biomass. Biomass production for bioenergy may cause large land use conversions, impact agricultural production, food prices, forest conservation, etc. The best solution is to use biomass that does not have agricultural or ecological value. Some of such unconventional sources of biomass are found within urban spaces. We employed Geographic Information System (GIS) and quantitative Life Cycle Assessment (LCA) methodologies to identify and estimate bioenergy potential of green roofs and other bioenergy options within urban areas. Net Energy Gain (NEG) and Energy Return on Energy Invested (EROEI) were used as indicators to assess the bioenergy potential of urban spaces within the Overijssel province of the Netherlands as a case study. Data regarding suitable areas were geometrically extracted from available GIS datasets, and used to estimate the biomass/bioenergy potential of different species with different yields per hectare, growing under different environmental conditions. We found that potential net-energy gain from built-up areas can meet 0.6–7.7% of the 2030 renewable energy targets of the province without conflicting with socio-ecological concerns, while also improving human habitat.