AbstractHarvesting corn stover can negatively impact soil chemical and physical properties, but less is known regarding its effects on soil microbiology. We evaluated the impact of corn (Zea mays L.) stover removal level (SRL) on soil microbial biomass (MB) and extracellular enzyme activities (EEAs) in irrigated, continuous corn located in eastern Nebraska. Soil fatty acid‐methyl esters (FAMEs) and EEA of eight soil enzymes were measured over 2 years, 0‐ to 20‐cm soil depth, under conventional disk tillage (CT) and no‐till (NT) under three SRLs. Soil chemical properties were measured as explanatory variables for changes in soil microbial communities and their enzymatic potential. Potential EEA of all enzymes decreased with stover removal and tillage. Soil bacterial biomass (BB) and fungal biomass (FB) decreased with SRL, while mycorrhizal biomass was unresponsive. Impacts of tillage on MB groups varied by year; however, FB was consistently higher under CT. At all growth stages, NT with all stover retained, a “soil conservation target” had distinct soil enzyme and bacterial FAME profiles compared to CT across all SRLs and to NT under high and sometimes moderate SRLs. Principal component analysis of soil properties was dominated by SRL with EEAs, BB, soil organic matter (SOM), water‐extractable carbon, and H3A‐extractable potassium being positively correlated to stover retention. Reduced input of microbial substrates also led to a loss of fine particulate organic matter, an indicator correlated to stable macroaggregation. Because NT partially ameliorated the negative impact of stover removal on BB, SOM fractions, and soil enzymes critical to nutrient cycling, pairing NT with stover removal may be more sustainable in the long term.