Temporal niche shifts can shape predator–prey interactions by enabling predator avoidance, enhancing feeding success, and reducing competition among predators. Using a community-based conservation approach, we investigated temporal niche partitioning of mammalian predators and prey across 12 long-term camera trap surveys in the Pacific slope and Talamanca Cordillera of Costa Rica. Temporal overlap and segregation were investigated between predator–prey and predator–predator pairs using overlap analysis, circular statistics, and relative abundance after accounting for differences in habitat, season, and human impact among sites. We made the assumption that predators select abundant prey and adjust their activity to maximize their temporal overlap, thus we predicted that abundant prey with high overlap would be preferred prey species for that predator. We also predicted that similar-sized pairs of predator species with the greatest potential for competitive interactions would have the highest temporal segregation. Our results supported the existence of temporal niche separation among the eight species of predators—the smaller Leopardus felids (ocelot, margay, oncilla) were primarily nocturnal, the largest felids (jaguar and puma) and coyote were cathemeral, and the smaller jaguarundi and tayra were mostly diurnal. Most prey species (67%) were primarily nocturnal versus diurnal or cathemeral (33%). Hierarchical clustering identified relationships among species with the most similar activity patterns. We discuss the primary prey and competitor species predicted for each of the eight predators. Contrary to our prediction, the activity pattern of similar-sized intraguild competitors overlapped more than dissimilar-sized competitors, suggesting that similar-sized predators are hunting the same prey at the same time. From this we conclude that prey availability is more important than competition in determining circadian activity patterns of Neotropical predators. Our results indicate the presence of a delicate balance of tropical food webs that may be disrupted by overhunting, leading to a depauperate community consisting of ubiquitous generalists and endangered specialists. With Central America a hotspot for hunting-induced “empty forests,” community-based conservation approaches may offer the best road to reduce illegal hunting and maintain the biodiversity and community structure of tropical forest systems.