Soil organic matter (SOM) is a major driver of key agroecosystem functions. Our objective was to examine the dynamics of organic matter in whole soil, particulate (POM; >53 µm size), and mineral-associated (MAOM) fractions under varying crop rotations and nutrient managements at two long-term experimental sites (Breton and Lethbridge). Soil samples were collected from simple (2 yr) and complex (5 or 6 yr) crop rotations at the 5–10 cm depth. We found associations between SOM pools versus microbial community and soil aggregation. Compared to cropped soils, an adjacent forest exhibited a significantly higher soil total organic carbon (TOC) and a shift in SOM fractions with substantially higher POM. However, the forest soil had the lowest microbial biomass C among all the assessed land use systems (P < 0.05), suggesting that other factors than the amount of labile SOM (i.e., POM-C) were controlling the microbial community. When contrasted to simple 2 yr rotations, the complex rotations including perennials and legumes significantly raised TOC and soil total nitrogen as well as the stable SOM fraction (i.e., MAOM-C and -N) consistently for both Breton and Lethbridge sites. Our findings highlight that varying land managements have profound feedbacks on soil quality as mediated by alterations in long-term SOM dynamics.