Context Implementing heterogeneous rural landscapes with high agricultural diversity and a substantial proportion of natural habitats has been proposed to ensure food production while reducing negative impacts on ecosystem services. However, evidence of an increased supply of ecosystem services (ES) in more heterogeneous landscapes remains limited, with no consensus. Objectives To evaluate the effect of the spatial cropland system’s diversity and landscape configuration on indicators of the supply of ES in agricultural landscapes of the Rio de la Plata Grasslands region. Methods We analyzed the relationship between indicators of ES supply and the heterogeneity of 1121 microwatersheds. We assessed the Ecosystem Services Supply Index (ESSI), the Hydrological Yield (HY), and the Absorbed Photosynthetically Active Radiation (APAR) in agricultural areas. We calculated the average grassland patch area, the structural and functional cropland diversity, the cropland percentage, and the grasslands’ juxtaposition to assess landscape heterogeneity. Results Microwatersheds with higher cropland functional diversity showed higher values for indicators of ES supply. They were positively related to the ESSI and APAR, and negatively with HY, indicating positive effects on Carbon gains and water regulation processes. In contrast, grasslands’ juxtaposition had opposite effects to those of cropland functional diversity, so the spatial segregation of grasslands favored the ES supply. Conclusions Functional cropland diversification and the segregation of natural grasslands improved proxies of ES and counteracted the negative effects of cropland amount. These findings contribute to the design of multifunctional landscapes and suggest that cropland functional diversity and grassland configuration should be considered in food production systems aimed at preserving ES supply.