Terrestrial ecosystems are often burned by both managed and wildfires. During a fire, the above-ground biomass that would typically accumulate and be decomposed as litter on the soil surface is combusted. Pyrogenic organic matter (PyOM) remaining after a fire is a considerably more recalcitrant organic input to the soil than fresh litter. The activity and community composition of soil organisms responsible for decomposition of organic matter and nutrient recycling in the soil could thus be affected by altered resource availability caused by frequent fires. We examined the effect of PyOM vs. fresh litter inputs to soil biota at both an annually burned and an infrequently burned tallgrass prairie site by tracing 13C- and 15N-labelled litter and PyOM into growing plant roots, microbial phospholipid fatty acids (PLFAs) and soil nematodes for 1 year. Litter-derived C or N was incorporated into microbes, nematodes and roots, while PyOM contributed minimally to soil microbes and plant roots and was not detectable in nematodes. Soil microbial and nematode abundances in the PyOM amended plots did not significantly differ from the bare soil plots, but nematodes were more abundant and microbial PLFAs less abundant in litter-amended plots. The annually burned site had less predator and omnivore nematodes, with increased abundance of microbial feeders and greater microbial uptake of litter C. An alteration of the soil food web structure caused by burning and the removal of the litter layer, along with the accumulation of non-microbially decomposed PyOM in the soil, contributes to the limitation of N cycling in frequently burned ecosystems. A Lay Summary is available for this article.