Noise produces multiple effects on ecosystems and it influences habitat use by vertebrates near roads. Thus, it may reduce the effectiveness of mitigation measures installed on roads to alleviate population fragmentation. This study analyses the effects of noise on the use by vertebrates of 19 underpasses at a motorway. It employs generalised linear models to test the effect of three noise indicators at the underpasses and in their vicinity on the crossing frequency of eight animal species. The results show that the road crossings are subjected to high and variable noise levels. Nevertheless, there is no consistent response to noise by vertebrates. This suggests that wildlife use of underpasses is determined more by habitat characteristics than by the levels of noise tolerated. The conclusion is that noise abatement measures on roads in areas of faunal sensitivity should focus on general noise reduction rather than on making individual crossing places quieter.