Strip-tillage is a conservation tillage technique that is being increasingly employed in vegetable crop production systems in the southeastern United States. Strip-tillage results in a delineated crop field, having properties of conventional tillage systems in the in-row locations and properties of no-till systems in the inter-row locations. Few studies have compared biological and physical soil properties and relationships at the whole-field scale in strip-tillage cropping systems with different input and crop rotation regimes. In this study we examined the spatial gradient of biological, physical, and chemical soil properties in the region spanning the field in-row to inter-row locations and compared results between systems utilizing different inputs (conventional fertilizer and pesticides vs. organic-approved inputs) and rotation regimes (continuous staked tomatoes vs. three-year vegetable rotation). Bulk density was significantly greater in the inter-row locations and in the rotation treatments, probably as a result of greater traffic relative to the continuous treatment. No differences in moisture content were determined between field locations or agricultural treatments. Inter-row locations contained greater soil C and N in organic compared to synthetic treatments, and in continuous compared to rotation treatments. Soil respiration was greatest in the inter-row and least in the in-row locations, and greater in treatments receiving organic inputs relative to synthetic inputs. No differences in soil N mineralization were observed. Microbial biomass C and N were greatest in inter-row and least in in-row locations, following total C availability. Principle components analysis further confirmed that greater values for biological parameters were obtained from inter-row locations, despite greater bulk density values.