Soil biological quality can affect key soil functions that support food production and environmental quality. The objective of this study was to determine the effects of management and time on soil biological quality in contrasting dryland cropping systems at eight locations in the North American Great Plains. Alternative (ALT) cropping systems were characterized by greater cropping intensity (less fallow), more diverse crop sequences, and/or reduced tillage than conventional (CON) cropping systems. Soil biological properties were assessed at depths of 0–7.5, 7.5–15, and 15–30 cm from 1999 to 2002 up to three times per year. Compared to CON, ALT cropping systems had greater microbial biomass and potentially mineralizable N. ALT cropping systems also had greater water stable aggregates in the surface 7.5 cm, but only at four locations. Total glomalin (TG), an organic fraction produced by fungi associated with aggregate stability, differed only at one location (Mandan), where the ALT cropping system had 27% more TG than the CON cropping system. Fatty acid methyl ester (FAME) profiles were highly location dependent, but total extracted FAME tended to be higher in ALT cropping systems. Soil biological properties fluctuated over time at all locations, possibly in response to weather, apparent changes in soil condition at sampling, and the presence or absence of fallow and/or legumes in rotation. Consequently, preplant and post-harvest sampling, when weather and soil conditions are most stable, is recommended for comparison of soil biological properties among management practices. Overall, ALT cropping systems enhanced soil function through: (1) improved retention and cycling of nutrients and (2) maintenance of biodiversity and habitat, implying improved agro-ecosystem performance over time.