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Activity pattern studies can help explain the coexistence of competing species. Between 2001 and 2017 we evaluated the activity pattern overlap of jaguar (Panthera onca), puma (Puma concolor), and their main prey, using camera traps at 17 Amazonian sites in the Greater Madidi-Tambopata Landscape. We used the Kernel density estimation to generate species activity patterns and the overlap between both cats. We then calculated the overlap coefficient (Δ) by carrying out 10,000 bootstraps (95%). Both cats were active 24 h a day. The puma has higher nocturnal activity (57%), whilst jaguar activity is split almost equally between night (53%) and day (47%). We did not find temporal segregation between jaguars and pumas, which showed similar activity patterns with a high overlapping coefficient (Δ4 = 0.84; 0.78–0.91). Also, we did not find significant differences between Male and Female activity patterns for both species (X2 = 0.50, gl = 1, P = 0.47). Moreover, both cats had significant overlap with the activity patterns of their main prey. Temporal segregation was not detected in any of our study sites within the Greater Madidi-Tambopata Landscape, suggesting that in this region, these predators employ other mechanisms to avoid competition.