Multifunctional agriculture provides noncommodity functions and services along with food, feed and bioenergy feedstocks, for example by preserving or promoting biodiversity, improving soil fertility, mitigating climate change and environmental degradation, and contributing to the socio‐economic viability of rural areas. Producing biomass for bioenergy from low‐input perennial species mixtures on marginal land has the potential to support biodiversity and soil carbon sequestration in synergy with greenhouse gas mitigation. We compared biomass production in species‐rich mixtures of perennial grasses, legumes and forbs with pure‐stand grasses and relatively species‐poor mixtures under different nitrogen fertilization regimes. Field experiments were performed on different types of marginal land, that is agricultural field margins and land with poor soil fertility, at four sites in southernmost and western Sweden. Biomass production was measured for three years in perennial grasses grown as pure stands, in legume‐grass mixtures, and legume‐grass‐forb mixtures across a species richness gradient. In unfertilized species‐rich mixtures, average biomass yields per experimental site and year were in the range from 3 to 9 metric ton DM ha⁻¹ yr⁻¹. While the most productive pure‐stand grasses fertilized with 60–120 kg N ha⁻¹ yr⁻¹ often produced higher biomass yields than unfertilized mixtures, these differences were generally smaller than the variations between years and sites. Calculations of climate impact using the harvested biomass for conversion to biogas as vehicle fuel showed that the average greenhouse gas emissions per energy unit were about 50% lower in unfertilized systems than in treatments fertilized with 100–120 kg N ha⁻¹ yr⁻¹. Our findings thereby show that unfertilized species‐rich perennial plant mixtures on marginal land provide resource‐efficient biomass production and contribute to the mitigation of climate change. Perennial species mixtures managed with low inputs thus promote synergies between productivity and biodiversity in the perspective of climate‐smart and multifunctional biomass production.