Tropical owls are one of the most threatened groups of birds. Studying the association between landscape features and owl assemblages is challenging due to the logistical difficulties of nocturnal fieldwork. We analyzed the association of landscape composition and spatial configuration of forest cover with the composition of the owl assemblages in a tropical rainforest in southeastern Mexico. During the 2014 reproductive season (April to June), we estimated owl abundance through auditory detection and playbacks at 60 survey points distributed in 12 landscapes (625 ha each) within a gradient of forest cover. We found that the owl community comprised six out of the 10 expected resident species. The landscape unit with 41% forest cover contained the most diverse assemblage. The owl species most susceptible to local extinction were specialists of interior areas of old-growth forest. The abundances of the Middle American Screech-Owl (Megascops guatemalae), Great Horned Owl (Bubo virginianus), and Black-and-white Owl (Ciccaba nigrolineata) were positively associated with the proportion of forest cover as well as with the average size of the patches, but negatively associated with environmental temperature. The abundances of the Barn Owl (Tyto alba) and Ferruginous Pygmy-Owl (Glaucidium brasilianum) were positively related to the amount of urban area in the landscape. The only species recorded along the entire gradient was the Mottled Owl (Ciccaba virgata), a species that is a generalist in habitat and diet; its abundance was positively related to the proportion of forest cover and the amount of water surface (lakes and rivers) in the landscape. Four resident species were not recorded: Crested Owl (Lophostrix cristata), Spectacled Owl (Pulsatrix perspicillata), Striped Owl (Pseudoscops clamator), and Central American Pygmy-Owl (Glaucidium griseiceps).