Amplification of a mitochondrial DNA fragment was used to compare the efficiency of five methods for extracting DNA from bat droppings. The Qiagen DNA Stool Kit, which yielded > 90% mtDNA amplification success, was chosen to extract DNA from 586 samples taken over two years in three French colonies of the lesser horseshoe bat (Rhinolophus hipposideros). Samples, for which mtDNA amplification was successful, were subject to the multiplex amplification of eight microsatellite loci. This resulted in > 95% amplification success over 12,592 PCRs. Allelic dropout (ADO) and false allele (FA) rates were low, and consequently, sample and locus quality indexes (QI) were high. These results demonstrate that large scale noninvasive studies of bat colonies are possible.