This study presents an investigation of Amiata donkey health and quality of milk for human consumption. Thirty-one lactating dairy jennies were examined. The following samples were collected: faecal samples from the rectum of animals for parasitological examination; cervical swabs for the detection of bacteria causing reproductive disorders; and blood samples for serological diagnosis of main zoonotic (Brucella spp., Leptospira spp.) and donkey abortion agents (Brucella spp., Leptospira spp., Salmonella abortus equi, Equine viral arterithis virus, Equine herpesvirus type 1). In addition, individual milk samples were collected and analysed for mastitis-causing pathogens and milk quality. Regarding animal health, we detected a high prevalence of strongyle parasites in donkeys. It is very important to tackle parasitic diseases correctly. Selective control programmes are preferable in order to reduce anthelmintic drug use. For dairy donkeys, withdrawal periods from anthelmintic drugs need to be carefully managed, in accordance with EU and national regulations. The isolation of Staphylococcus aureus in milk highlights the importance of preventing contamination during milking, by adopting appropriate hygiene and safety practices at a farm level. Lysozyme activity was high compared to cow’s milk, contributing to the inhibitory activity against certain bacteria. Donkey milk was characterised by high lactose content, low caseins, low fat, higher levels of unsaturated fatty acids compared to ruminant milks. Unsaturated fatty acids and omega 3 fatty acids in particular have become known for their beneficial health effect, which is favourable for human diet. These characteristics make it suitable for infants and children affected by food intolerance/allergies to bovine milk proteins and multiple food allergies as well as for adults with dyslipidemias. It is also recommended to prevent cardiovascular diseases.