Jaguars, Panthera onca (Linnaeus, 1758), and pumas, Puma concolor (Linnaeus, 1771) are the largest felids in the neotropics. Both can overlap in niche axes (time, space and prey), and are therefore potentially competing species. Segregation mechanisms presented by a low overlap in one of these axes of niche can facilitate the coexistence. Our aim was to analyze jaguar and puma temporal and spatial overlap for understanding their segregation mechanisms. Between 2015 and 2017, twenty-six camera trap stations were located in five habitat types of El Cielo Biosphere Reserve (ECBR) in northeastern Mexico. Temporal activity was analyzed using circular statistics and time overlap analysis. Spatial overlap was calculated with the Pianka index and a selectivity habitat analysis. Our results showed that jaguars and pumas were nocturnal and that the temporal overlap was high (∆4 = 0.77). We found an intermediate spatial overlap (Pianka index = 0.61). Jaguars were more selective and preferred the deciduous forest. In comparison, pumas preferred oak-pine forest, but also used oak and deciduous forest. Our results indicate that spatial segregation best explains the coexistence of jaguars and pumas in our study area, probably due to both habitat diversity in the reserve and the generalist habits of the puma.