Knowledge on the effects of land use on community composition and species abundance is crucial for designing realistic conservation strategies, particularly in highly dynamic systems such as Mediterranean agricultural mosaics that are subjected to intensive cultivation. We investigated these effects on the nocturnal bird species occurring in the study area (Stone Curlew Burhinus oedicnemus, Red-necked Nightjar Caprimulgus ruflcollis, Barn Owl Tyto alba, Eurasian Scops Owl Otus scops, Little Owl Athene noctua, Tawny Owl Strix aluco, Long-eared Owl Asio otus, Short-eared Owl Asio flammeus and Eagle Owl Bubo bubo) across an agricultural-natural habitat mosaic in Central Spain for three consecutive years. Shares of vineyards, scrubland, herbaceous cropland, water bodies, and roads significantly affected the composition of the nocturnal bird community. Herbaceous cropland and olive groves, which covered 50% of the study area, proved to be neutral for all species. Remnant patches of natural and semi-natural scrubland (around 10% of the study area) and water bodies (only 1.5% of the study area) showed a positive effect on Eagle Owls, Eurasian Scops Owls, Long-eared Owls, and Red-necked Nightjars. Vineyard (35% of the study area) had a negative influence on Eagle Owls, Long-eared Owls, and Eurasian Scops Owls. Our results indicate, first, that the relative extent of land use types was apparently not related with the presence of nocturnal bird species and, second, that natural scrublands and water bodies are key habitats for assuring the persistence of nocturnal birds in agricultural Mediterranean landscapes. Current land planning focused toward land use intensification will likely increase the areas of habitats that are neutral or have adverse effects on nocturnal birds.