We aimed to evaluate the temporal and spatial use of waterholes by ocelots in Calakmul Biosphere Reserve (CBR), Mexico. From 2014 to 2017, we monitored 11 waterholes with camera traps. We compared diel activity patterns with circular statistics depending on waterholes’ level of human intensity and distance to the Calakmul road, seasonality, and sex. We identified 40 different ocelots. Four waterholes were the most important ones, being 2 of them close to the road. Individuals took on average 19 days to return to waterholes. The diel activity of ocelots was 63.67% nocturnal, 20.70% crepuscular and 15.60% diurnal, and they were more diurnal in waterholes distant from the road. Their activity pattern was bimodal and it did not change between any of the categories tested. This is the first study to determine the spatial and temporal activity of ocelots in waterholes of Mexico. Ocelots are mainly nocturnal, and this pattern is conserved throughout CBR, however, they are able to adjust slightly their activity depending on extrinsic factors, such as an increased human presence. In the Calakmul region, all waterholes are crucial, and we particularly emphasize the conservation of the most important waterholes for ocelots, especially the ones close to the road.