Soil-borne oomycetes include devastating plant pathogens that cause substantial losses in the agricultural sector. To better manage this important group of pathogens, it is critical to understand how they respond to common agricultural practices, such as tillage and crop rotation. Here, a long-term field experiment was established using a split-plot design with tillage as the main plot factor (conventional tillage (CT) vs. no till (NT), two levels) and rotation as the subplot factor (monocultures of soybean, corn, or wheat, and corn–soybean–wheat rotation, four levels). Post-harvest soil oomycete communities were characterized over three consecutive years (2016–2018) by metabarcoding the Internal Transcribed Spacer 1 (ITS1) region. The community contained 292 amplicon sequence variants (ASVs) and was dominated byGlobisporangiumspp. (85.1% in abundance, 203 ASV) andPythiumspp. (10.4%, 51 ASV). NT decreased diversity and community compositional structure heterogeneity, while crop rotation only affected the community structure under CT. The interaction effects of tillage and rotation on most oomycetes species accentuated the complexity of managing these pathogens. Soil and crop health represented by soybean seedling vitality was lowest in soils under CT cultivating soybean or corn, while the grain yield of the three crops responded differently to tillage and crop rotation regimes.