Eight different 3-year cropping systems, consisting of soybean-canola, soybean-barley, sweet corn-canola, sweet corn-soybean, green bean-sweet corn, canola-sweet corn, barley-clover, and continuous potato (non-rotation control) followed by potato as the third crop in all systems, were established in replicated field plots with two rotation entry points in Presque Isle, ME, in 1998. Cropping system effects on soil microbial community characteristics based on culturable soil microbial populations, single carbon source substrate utilization (SU) profiles, and whole-soil fatty acid methyl ester (FAME) profiles were evaluated in association with the development of soilborne diseases of potato in the 2000 and 2001 field seasons. Soil populations of culturable bacteria and overall microbial activity were highest following barley, canola, and sweet corn crops, and lowest following continuous potato. The SU profiles derived from BIOLOG ECO plates indicated higher substrate richness and diversity and greater utilization of certain carbohydrates, carboxylic acids, and amino acids associated with barley, canola, and some sweet corn rotations, indicating distinct differences in functional attributes of microbial communities among cropping systems. Soil FAME profiles also demonstrated distinct differences among cropping systems in their relative composition of fatty acid types and classes, representing structural attributes of microbial communities. Fatty acids most responsible for differentiation among cropping systems included 12:0, 16:1 ω5c, 16:1 ω7c, 18:1 ω9c, and 18:2ω6c. Based on FAME biomarkers, barley rotations resulted in higher fungi-to-bacteria ratios, sweet corn resulted in greater mycorrhizae populations, and continuous potato produced the lowest amounts of these and other biomarker traits. Incidence and severity of stem and stolon canker and black scurf of potato, caused by Rhizoctonia solani, were reduced for most rotations relative to the continuous potato control. Potato crops following canola, barley, or sweet corn provided the lowest levels of Rhizoctonia disease and best tuber quality, whereas potato crops following clover or soybean resulted in disease problems in some years. Both rotation crop and cropping sequence were important in shaping the microbial characteristics, soilborne disease, and tuber qualities. Several microbial parameters, including microbial populations and SU and FAME profile characteristics, were correlated with potato disease or yield measurements in one or both harvest years. In this study, we have demonstrated distinctive effects of specific rotation crops and cropping sequences on microbial communities and have begun to relate the implications of these changes to crop health and productivity.