Water availability is a primary limiting factor facing agricultural systems in most semi-arid regions across the world. This study is part of a larger long-term project to develop and evaluate integrated crop and livestock systems in order to reduce dependence on underground water sources by optimizing cotton (Gossypium hirsutum) production in the Texas High Plains of U.S. Selected microbial, chemical and biochemical properties were studied (between 7 and 10 years) in a clay loam soil (fine, mixed, thermic Torrertic Paleustolls) under continuous cotton compared to an integrated cropping-livestock system that included cotton, forage, and Angus-cross-stocker beef steers (initial body weight 249 kg). For the integrated system, steers grazed in sequence a perennial warm-season grass ‘WW-B. Dahl’ old world bluestem (Bothriochloa bladhii) paddock, and then rye (Secale cereale L.) and wheat (Triticum aestivum L.) grown in two paddocks (stages) of a rotation with cotton. Our previous studies after 5 years showed greater microbial biomass C (MBC) in perennial pasture (193 mg kg⁻¹ soil) and the rotation when sampled under rye or cotton (average of 237 mg kg⁻¹ soil) compared to continuous cotton (124 mg kg⁻¹ soil) at 0–5 cm. After 7 years, MBC became significantly higher in the rotation independent of the crop compared to continuous cotton in this study. At the end of 10 years, total C was higher in both the rotation and pasture of the integrated cropping-livestock system (average across grazing treatments: 17.3 g kg⁻¹ soil) compared to continuous cotton (11.4 g C kg⁻¹ soil). Soil MBC and several enzyme activities were higher under non-grazed areas compared to grazed areas within the integrated cropping-livestock system in some samplings. Microbial community structure of pasture soil showed higher FAME indicators for G− (i.e., a17:0 and cy19:0) and actinomycetes (i.e., 10Me17:0) under grazed areas compared to non-grazed areas. Microbial community structure of pasture soil showed higher fungal populations compared to continuous cotton. The rotation showed intermediate sum of bacterial FAME indicators among systems (continuous cotton > rotation > pasture) and a tendency for numerically slightly higher fungi:bacterial ratios compared to continuous cotton. This study demonstrated increases in microbial biomass and enzyme activities of C-, N-, P- and S-cycling within an integrated cropping-livestock system that may represent positive changes in soil functioning compared to continuous cotton.