Abstract Integrated crop and livestock (ICL) agroecosystems are characterized by a mixture of perennial or annual vegetation grazed by livestock and annual harvested crops. Compared to annual crops, ICLs hold the potential to enhance soil organic matter (OM) inputs, carbon sequestration, nutrient cycling, and water conservation. Soil fungi play an essential role in the transformation of OM and nutrients and soil structure stabilization; however their specific role in OM transformations in ICL agroecosystems has not been studied. This study evaluated mycorrhizal and saprophytic fungal populations (via fatty acid methyl ester profiles; FAME) and saprophytic fungal functionality (via FungiLog analysis) under two ICL agroecosystems and a continuous cotton (Gossypium hirsutum L.) system in the Southern High Plains of the U.S. The first ICL system included non-irrigated perennial native grasses, an annual cotton and foxtail millet (Setaria italica) rotation and deficit-irrigated ‘WW-B. Dahl’ old world bluestem (Bothriochloa bladhii; OWB). The second ICL agroecosystem consisted of deficit-irrigated OWB and bermudagrass (Cynodon dactylon). The effect of grazing by cattle was evaluated via grazing exclusion areas. Abundance of saprophytic fungal FAMEs (10–26% of total FAMEs) and mycorrhizal FAMEs (2–24% of total FAMEs) were higher under ICLs compared to the continuous-cotton system at 0–5 cm. Overall, vegetation impacted the distribution of the fungal FAME markers, whereas the fungal saprophytic functionality was more sensitive to grazing. Perennial vegetation of ICLs was associated with an increase in fungal markers (saprophytic and mycorrhizal) as well as increased soil OM content. Greater utilization of multiple C sources and increased saprophytic fungal functional indices were found under cotton, non-grazed perennial vegetation (with exception of bermudagrass) and the rotation under millet. Among the grazed perennial vegetation, bermudagrass showed the highest fungal FAMEs abundance and functional diversity values. These fungal improvements were also reflected in the highest OM content under this grass, potentially indicating improved sustainability under the OWB and bermudagrass agroecosystem.