Acoustic surveys represent a powerful tool to assess bat distribution and habitat preferences and are widely applicable to monitoring and conservation schemes. However, their correct application requires the development of robust and reliable identification procedures. Little information is available on the bats of Madeira Island (Portugal), particularly their ecological requirements, distribution and population trends, and proper guidelines for their monitoring and conservation have yet to be defined. In this study, we present the first analysis of echolocation calls from species occurring on the island to provide a tool for bat identification during acoustic surveys. Seven hundred and ninety one time-expanded recordings of search phase echolocation calls in cluttered and uncluttered habitats of the Macaronesia endemic pipistrelle Pipistrellus maderensis, Madeira Leisler’s bat Nyctalus leisleri verrucosus and grey long-eared bat Plecotus austriacus were analysed and an acoustic repertoire with their temporal and spectral features is described. Pipistrellus maderensis was easily identified based only on peak frequency while Plecotus austriacus and N. leisleri verrucosus showed frequency overlap in cluttered backgrounds. Discriminant function analysis was applied to calls of Plecotus austriacus and N. l. verrucosus resulting in a correct overall classification of 96.7% of calls, with a function based on start frequency, end frequency, duration and interpulse interval. This work provides the first description of Pipistrellus maderensis and N. 1. verrucosus echolocation calls and offers a basis for future bat surveys in order to encourage the development of locally customized conservation strategies.