ABSTRACT: Microbial biomass is a driving force in the dynamics of soil organic matter, and microbial activity is an indicator of soil quality in agroecosystems, reflecting changes in management practices and environmental conditions. We evaluated the effect of monoculture and intercropped winter cover crops on soil chemical attributes, microbial biomass carbon (MBC), basal respiration (BR), metabolic quotient (qCO2), urease, β-glucosidase, and fluorescein diacetate (FDA) hydrolysis activity, as well as onion yield in a no-tillage system. Soil is a Typic Humudept, and treatments were control with spontaneous vegetation, barley, rye, oilseed radish (OR), OR + rye, and OR + barley. The soil was sampled (0-10 cm) five times between June and December. There were no differences among treatments for MBC and BR, and the highest values for those attributes occurred in June, when cover plants were in their initial stage. Although, qCO2 was not affected by any treatment, it varied among sampling periods, ranging from 0.62 to 10 µg C-CO2 mg-1 MBC h-1, indicating a low- or no stress environment. Cover crops had little influence on enzyme activity, but FDA was lowered in areas with single crops of barley and rye. Average onion yield in cover crops treatments was 13.01 (Mg ha-1), 30-40% higher than in the control treatment.