There is an increased interest in using donkey’s milk as a source of human nutrition. The large amounts of antimicrobial components and defense factors present in donkey’s milk provide protection from microbial infections and distinguish donkey’s milk from the milk of other mammals. However, the microbiota in donkey’s milk has so far been poorly characterized, specifically with regard to the lactic acid bacteria (LAB). This study has identified cultivable, acidifying and thermoduric LAB that could be used to develop starter cultures. This is the first study to investigate the culturable LAB microbiota present in donkey’s milk.