The Atlantic Forest is one of the most disturbed Brazilian biomes, with 183 out of 298 species of mammals occurring in the state of Rio de Janeiro. In this study, we aimed to characterize the diversity, community structure, and habitat use of small mammals in the FIOCRUZ Atlantic Forest Campus (CFMA), including areas of Pedra Branca State Park (PBSP, subunit Pau da Fome), state of Rio de Janeiro. We also compared species diversity and composition between two moments 15 years apart (2001 and 2012–2015) and analyzed the population dynamics of the marsupial Didelphis aurita (Wied-Neuwied, 1826). Small mammal captures were made in different habitats: sylvatic-urban interface areas near human dwellings, disturbed forest, and preserved forest areas. Five marsupial species and four rodent species were captured in both periods. There was a reduction in species richness and β diversity between the two periods, indicating that disturbances in the environment over the years may have affected the small mammal community structure. The most altered environment showed the greatest species richness and abundance, while the forest areas showed the smallest values, which may be explained by the loss of mammal species, mainly specialist species in forested areas. We identified three groups of species according to habitat preferences: one related to environments with a higher density of vegetation in upper strata – Marmosa paraguayana (Tate, 1931) and Monodelphis americana (Müller, 1776), another related to a higher density in lower forest strata – Akodon cursor (Winge, 1887), and another with no association with the investigated habitat variables – D. aurita and Oligoryzomys nigripes (Olfers, 1818). The small mammal community structure showed a low level of nestedness in both sampling periods. This study is the first report to evaluate the community structure of small mammals in the sylvatic-urban interface area of Pedra Branca State Park, the largest forest reserve within an urban area in Brazil. The surveys indicate that the small mammal diversity was low in both sampling periods and in both areas, and a species loss in the Pau da Fome locality was observed, despite it is a conservation unit. The greater species abundance and richness in the most disturbed areas suggest an increase of factors favoring the occurrence of synanthropic and opportunistic species.