In the last century bat populations significantly declined mainly due to habitat fragmentation and degradation. As management-dependent species, bats need appropriate monitoring programs for the implementation of sound conservation strategies. However, bats’ small size, high mobility, elusiveness and nocturnal lifestyle make them difficult to survey. Non-Invasive Genetic Sampling (NIGS) may offer safe and cost-effective solutions, but requires well-planned sampling strategies, informative molecular markers and reliable laboratory protocols. Here we developed a NIGS protocol for species and individual identification of three mouse-eared bats, the Geoffroy’s bat (Myotis emarginatus), the long-fingered bat (Myotis capaccinii) and the Daubenton’s bat (Myotis daubentonii). Species identification was accomplished by mitochondrial (mt) DNA sequencing of reference tissue (n=49) and droppings (n=285) from Central-Northern Italy. In addition, we optimized a multiplex panel of seven microsatellites suitable for species and individual identification of the three species from droppings. We obtained a good success with mtDNA sequencing (245/285; 86%) and microsatellite genotyping (129/245; 53%). All microsatellites were successfully amplified with low error rates, and were polymorphic in the three Myotis species, with probabilities of identity 6 0.001 and observed heterozygosities of H_o=0.48 in M. emarginatus, 0.62 in M. capaccinii and 0.71 in M. daubentonii. Our protocol represents a useful tool for population genetic studies on mouse-eared bats that could likely be extended to other bat species and provide useful information to implement effective conservation plans.