This study investigated the effect of road and highway proximity on the occurrence of bird species in isolated secondary pine forest fragments in an agricultural matrix. We assessed the effects of road proximity separately for three different groups of bird species in order to detect ecological responses related to species biology. Bird occurrence was mainly related to fragment size but also to vegetation structure and fragment connectivity. When corrected for patch characteristics, we found that fragments up to 2 km away from a main highway contained less forest species, both generalists and specialists than fragments more distantly located. This pattern was independent of the side of the highway analysed. Considering each bird species separately, we found consistent lower occurrence probabilities near the highway in 50% of forest species. This difference was not found for ubiquitous species that were to some extent positively influenced by proximity of other major roads. Our results suggest that highway proximity, but not that of other major roads, decrease occupancy probability of forest birds in isolated forest fragments, thus reducing probability of metapopulation persistence. Although low habitat quality as a result of noise disturbance is likely to account for some of the results, the long distance effect detected strongly suggests that other factors rather than direct traffic disturbance alone are involved in lower bird occurrences near the highway. Decreased connectivity among forest fragments associated to avoidance of areas near the highway is an alternative explanation to the results obtained. We suggest that these findings should be included in strategic environmental impact assessment studies conducted to determine the ecological impact of large transport infrastructures across highly fragmented landscapes.