Soil fungi perform a variety of ecosystem functions that are crucial to maintaining agroecosystem sustainability including aggregate stability and soil carbon storage.  The purpose of this study was to compare soil fungal communities and functional diversity in integrated crop and livestock (ICL) systems with cotton monoculture systems. Soil samples (0-5 and 5-20 cm) were collected from three ICL systems and two cotton cropping (CTN) systems in the semi-arid Texas High Plains region. Within two ICLs, samples also were collected from exclosures to determine the effect of grazing. The relative abundance of soil fungi and fungal:bacteria ratios were determined using fatty acid methyl esters (EL-FAME). System activity (SA) and system richness (SR), calculated from fungal substrate-utilization assays (FungiLog), were used to assess fungal functional diversity. The arbuscular mycorrhizal FAME biomarker (16:1w5c) was greater in the 5-20 cm samples than at 0-5 cm. All other FAME fungal biomarkers were lower with depth. The mol% of AMF was greater under ICLs compared to the cotton systems but this pattern was reversed with the 18:2ω6,9 fungal biomarker. Agricultural system vegetation, soil depth and grazing affected SA and SR values. Unexpectedly, the 18:2w6,9 fungal:bacterial ratio was higher in CTN compared to ICL systems at both depths, while other fungal biomarkers (18:3w6,9,12) failed to differentiate between them. The two cotton systems had greater SA and SR values compared to two of the ICLs. Due to the complexity of different aboveground vegetation types among systems (e.g., one ICL was comprised of six different species), interpretation of FAME and FungiLog data at the vegetation level provided greater resolution of fungal differences within systems. Our results showed that alternative agricultural management practices impact fungal relative abundance and functioning, illustrating the importance of fungal analyses in agroecosystem research.