Changes in soil microbiotic properties such as microbial biomass and community structure in response to alternative management systems are driven by microbial substrate quality and substrate utilization. We evaluated irrigated crop and forage production in two separate four-year experiments for differences in microbial substrate quality, microbial biomass and community structure, and microbial substrate utilization under conventional, organic, and reduced-tillage management systems. The six different management systems were imposed on fields previously under long-term, intensively tilled maize production. Soils under crop and forage production responded to conversion from monocropping to crop rotation, as well as to the three different management systems, but in different ways. Under crop production, four years of organic management resulted in the highest soil organic C (SOC) and microbial biomass concentrations, while under forage production, reduced-tillage management most effectively increased SOC and microbial biomass. There were significant increases in relative abundance of bacteria, fungi, and protozoa, with two- to 36-fold increases in biomarker phospholipid fatty acids (PLFAs). Under crop production, dissolved organic C (DOC) content was higher under organic management than under reduced-tillage and conventional management. Perennial legume crops and organic soil amendments in the organic crop rotation system apparently favored greater soil microbial substrate availability, as well as more microbial biomass compared with other management systems that had fewer legume crops in rotation and synthetic fertilizer applications. Among the forage production management systems with equivalent crop rotations, reduced-tillage management had higher microbial substrate availability and greater microbial biomass than other management systems. Combined crop rotation, tillage management, soil amendments, and legume crops in rotations considerably influenced soil microbiotic properties. More research will expand our understanding of combined effects of these alternatives on feedbacks between soil microbiotic properties and SOC accrual.