Small mammal assemblages from South America provide a unique opportunity to measure coexistence and niche partitioning between marsupials and placentals. We tested how these two major clades partition environmental resources by comparing stable isotopic ratios of similar sized Didelphidae and Sigmodontinae in four Brazilian biomes: Pampas grassland, Pantanal wetland, Cerrado woodland savanna, and Atlantic Forest. Generally, didelphid isotopic niche follows a scaling law, because we found an association between δ¹⁵N enrichment and body mass. Sigmodontines that primarily partition the environment via forest strata showed a greater intake of C₄ or/and crassulacean acid metabolism (CAM) plants than didelphids, as reflected by their wider trophic niche. Values of δ¹³C were highest in savannas and grasslands (Cerrado and Pampas biomes), and values of δ¹⁵N were highest in the Atlantic Forest (in sigmodontines) and Pampas (in didelphids). While assessing patterns between the two major Brazilian biomes (Atlantic Forest and Cerrado), we found evidence of a broader trophic niche for both clades in the Cerrado. In the Atlantic Forest, niche occupation by Didelphidae was completely enclosed within the Sigmodontinae trophic niche. Both clades showed less overlap in the Cerrado, a less productive environment. Our results highlight the importance of a comparative framework and the use of stable isotopes for testing ecological questions related to how small mammalian communities partition their niche.