The gaseous N losses mediated by soil denitrifiers are generally inferred by measuring N2 O fluxes, but should include associated N2 emissions, which may be affected by abiotic soil characteristics and biotic interactions. Soil fauna, particularly anecic earthworms and euedaphic collembola, alter the activity of denitrifiers, creating hotspots for denitrification. These soil fauna are abundant in perennial agroecosystems intended to contribute to more sustainable production of bioenergy. Two microcosm experiments were designed to evaluate gaseous N emissions from a silty loam and a sandy soil, both provided with litter from the bioenergy crop Silphium perfoliatum (cup-plant) and inoculated with an anecic earthworm (Lumbricus terrestris), which was added alone or together with an euedaphic collembola (Folsomia candida). In experiment 1, litter-derived N flux was determined by adding 15 N-labelled litter, followed by mass spectrometric analysis of N2 and N2 O isotopologues. In experiment 2, the δ18 O values and 15 N site preference of N2 O were determined by isotope ratio mass spectrometry to reveal underlying N2 O formation pathways. Lumbricus terrestris significantly increased litter-derived N2 emissions in the loamy soil, from 174.5 to 1019.3μgN2 -Nkg-1 soil, but not in the sandy soil (non-significant change from 944.7 to 1054.7μgN2 -Nkg-1 soil). Earthworm feeding on plant litter resulted in elevated N2 O emissions in both soils, derived mainly from turnover of the soil mineral N pool during denitrification. Folsomia candida did not affect N losses but showed a tendency to redirect N2 O formation pathways from fungal to bacterial denitrification. The N2 O/(N2 +N2 O) product ratio was predominantly affected by abiotic soil characteristics (loamy soil: 0.14, sandy soil: 0.26). When feeding on S. perfoliatum litter, the anecic L. terrestris, but not the euedaphic F. candida, has the potential to cause substantial N losses. Biotic interactions between the species are not influential, but abiotic soil characteristics have an effect. The coarse-textured sandy soil had lower gaseous N losses attributable to anecic earthworms. Copyright © 2016 John Wiley & Sons, Ltd.