Given the phylogenetic proximity and similar morphology of opossums (Didelphis spp.), they are good models to study factors that facilitate or impede coexistence of syntopic species and to better understand how landscape changes affect species distributions and habitat use. For this purpose, we used single-species and two-species occupancy models using records of D. albiventris, considered the dominant species, and D. aurita from a camera-trap survey conducted in an agricultural and conservation landscape in the Atlantic Forest of Argentina. We evaluated which factors determined the probability of species occurrence: habitat preferences or interspecific relationships. We also estimated the overlap in daily activity patterns between species, and evaluated changes in D. aurita activity in response to the occupancy probability of D. albiventris. Didelphis aurita had higher occupancy probabilities in the continuous native forest, whereas D. albiventris had higher occupancy probabilities in fragmented forests with less complex and more open vegetation structure, and greater proximity to buildings. Both opossums were almost absent in pine plantations. Results of the co-occurrence models and the overlap in diel activity suggest that D. aurita is not avoiding D. albiventris. Occurrences of these two opossums most probably reflect different adaptations by each species to different habitats, and competitive interactions seem to play a minor role in shaping their current distributions. Didelphis albiventris may be replacing D. aurita mainly as a result of changing environmental conditions, which become unfavorable to the latter but promote the creation of new habitat for the former.