Aims The integrated crop-livestock systems (ICLS) under no-tillage improves soil fertility of the Brazilian Cerrado. We aimed to evaluate the effect of different grass-legume intercropping compared to single grass cultivation in the off-season of an ICLS on (i) soil organic carbon (C) and nitrogen (N) pools, (ii) soil microbial biomass and activity, (iii) soil enzymatic activity, and (iv) soybean grain yield in succession. Methods The field study was conducted in an on-farm experiment in 2016/17 and 2017/18 cropping seasons. The soybean was cultivated in the summer season, with the subsequent treatments in the off-season, using two grasses (Urochloa ruziziensis and U. brizantha), single or intercropped with Cowpea (Vigna unguiculata) or Pigeon pea (Cajanus cajan). We evaluated soil organic C and N pools, microbial biomass and activity, enzyme activity, and soybean yield. Results Cowpea intercropping yielded 24% more soybeans than grasses single cropped. There was a higher microbial biomass and activity, and enzymatic activity in the soil under grass-legume intercropping. In addition to the lower basal respiration and microbial metabolic quotient (qCO2), the greater microbial quotient (qMIC) and microbial biomass C indicate a higher soil microbial C utilization efficiency under grass-legume intercropping. The soil total organic C and N stocks increased under Pigeon pea intercropping by 16% and 27%, respectively, compared to single grasses. Conclusions Grass-legume intercropping in the pasture phase of ICLS is an additional tool to maximize soybean yields in the short term. The intercropping effects on soybean yield were directly related to soil quality improvements through soil biological and biochemical properties.