Species co-existence depends on how organisms utilize their environment and resources. When two sympatric species are similar in some ecological requirements, their coexistence may arise from differences in resource use over time and/or space. Interactions among coexisting marsupials remain poorly understood, especially in the Neotropics. Here we combine spatial niche measurements, individual-resource networks, and isotopic niche approaches, to investigate the ecological strategies used by the Neotropical marsupials Didelphis aurita and Metachirus nudicaudatus to co-occur in an area of Serra do Mar State Park (southeast of Brazil). Both individual-resource networks and isotopic niche approaches indicate similar patterns of omnivory for both species. Isotopic analysis showed the species' trophic niche to be similar, with 52% of overlap, and no differences between proportional contributions of each resource to their diets. Moreover, individual-resource network analysis found no evidence of diet nestedness or segregation. The trophic niche overlap observed was associated with spatial segregation between species. Despite using the same area over the year, D. aurita and M. nudicaudatus exhibited spatial segregation among seasons. These results illustrate that the detection of spatial segregation is scale-dependent and must be carefully considered. In conclusion, our findings provide a new perspective on the ecology of these two Neotropical marsupials by illustrating how the association of distinct but complementary methods can be applied to reach a more complete understanding of resource partitioning and species coexistence.