BackgroundCrop rotation is an agronomic practice that is known to enhance productivity and yield, and decrease pest and disease pressure. Economic and other factors have increased the frequency of certain crops, including canola, with unknown effects on the below ground microbial communities that impact plant health and performance. This study investigated the effect of 12 years of crop rotation including canola-wheat; canola-pea-barley; and unrotated canola across three geographic sites in Western Canada with diverse soil types and environmental conditions. To provide data on mature, established crop rotation strategies, root exudate profiles, soil nutrient fluxes, and bacterial and fungal microbial community profiles were determined at the flowering stage in the final two (canola) years of the 12-year rotations.ResultsAfter 12 years of rotation, nutrient fluxes were affected in the soil in an inconsistent manner, with K, NO3, Mg, Ca, P, and Fe fluxes variably impacted by rotation depending on the year and site of sampling. As expected, rotation positively influenced yield and oil content, and decreased disease pressure fromLeptosphaeriaandAlternaria. In two of the three sites, root exudate profiles were significantly influenced by crop rotation. Bacterial soil, root, and rhizosphere communities were less impacted by crop rotation than the fungal communities. Fungal sequences that were associated with specific rotation strategies were identified in the bulk soil, and included known fungal pathogens in the canola-only strategy. Two closely related fungal sequences identified asOlpidium brassicaewere extremely abundant at all sites in both years. One of these sequences was observed uniquely at a single site and was significantly associated with monocropped canola; moreover, its abundance correlated negatively with yield in both years.ConclusionsLong-term canola monoculture affected root exudate profiles and soil nutrient fluxes differently in the three geographic locations. Bacterial communities were less impacted by rotation compared to the fungal communities, which consistently exhibited changes in composition in all ecological niches at all sites, in both years. Fungal sequences identified asO. brassicaewere highly abundant at all sites, one of which was strongly associated with canola monoculture. Soil management decisions should include consideration of the effects on the microbial ecosystems associated with the plants in order to inform best management practices.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40793-023-00495-9.