The increasing road networks threaten ecosystems by direct effects such as increased mortality due to collision with vehicles and by various indirect effects leading to road avoidance. We censused Tawny Owls Strix aluco and Little Owls Athene noctua in 2005, 2007 and 2009 in a rural landscape in Southern Portugal in order to study the effects of roads and habitat characteristics on Tawny Owl density and Little Owl presence. The presence of both owl species in the 70 census locations was coherent among years. Our results showed that Tawny Owl density near major roads was lower, with the negative effects extending possibly up to 2 km. The probability of Little Owl presence was also negatively affected by the proximity to major roads. The negative effects of roads were significant even considering habitat preferences and spatial autocorrelation, which had the most marked effect on the density or presence of both owls. The reduced occupancy by Tawny Owls and Little Owls of habitats near major roads may be caused by several factors, including increased mortality, disturbance caused by high traffic density, and increased fragmentation. Traffic noise in particular may affect intra-specific communication and hunting efficiency. Consequently, habitat near roads may represent lower-quality territories for owls.