Abstract Circadian processes are key drivers of animal behaviour, influencing patterns of activity, resource partitioning and competition avoidance. Studies evaluating circadian changes on the structure of marine assemblages are lacking, especially for reef fish. Evaluating the changes imposed by the day–night cycle on the structure and functioning of these assemblages is of critical importance to understand the differences between the diurnal and nocturnal components, as well as the resources they exploit, to better model and manage them. Here, we assessed the fish fauna using an underwater visual census conducted at the same sites during both day and night on a subtropical rocky reef in the Southwestern Atlantic (SWA), aiming to evaluate differences in the community and functional structure of these assemblages. A total of 242 transects were conducted across three sites, with 104 during the day and 138 at night. Fish richness, density, biomass and functional richness were higher in the diurnal period, whereas nocturnal assemblages exhibited higher taxonomic distinctiveness and functional divergence. Both richness and density of nocturnal assemblage, as well as richness and taxonomic distinctness index of the diurnal assemblage, were influenced by depth. The species in each assemblage exhibited different combinations of traits. As expected, diurnal assemblages were richer and occupied a larger proportion of the multidimensional trait space. Body size was comparatively larger for the diurnal assemblage, evidencing niche partitioning based on ontogeny. These results indicate significant differences in the structure of diurnal and nocturnal reef fish assemblages, as expected, driven by resource use, space use and predator avoidance. Fish play a central role in many key processes within reef systems, in addition to their importance for human nutrition and cultural services. Therefore, understanding the components and diversity of nocturnal assemblages is essential to address the significant knowledge gap to understand their role in reef energetics and demand proper management.