Toxoplasmosis is one of the five parasitic diseases considered as a priority for public health action. The consumption of raw milk products represents a possible risk, in particular for certain categories of people. The aim of this study was to evaluate the possible effects of Toxoplasma gondii on milk yield and quality in sero-positive animals with parasitemia. Eighteen healthy lactating Amiata jennies, between 90 and 180 days were included in the study. Four donkeys scored positive for immunofluorescent antibody test (IFAT), and each IFAT positive donkey presented parasitic DNA both in the blood and milk. No significant differences were found between milk yield in PCR-positive donkeys compared with the negative cases, however the former tended to have a greater production. Milk quality in the positive donkeys showed a significantly lower percentage of casein (0.72% v. 0.81%) and ash (0.32% v. 0.37%). Positive cases had a highly significant larger average diameter of globules (2.35 µm) and fewer globules/ml (2.39×108). Somatic cell and bacterial counts were normal and in agreement with the literature. Toxoplasma gondii did not seem to present clinical forms in lactating jennies. Further in vivo studies are needed to further assess the risk of T. gondii transmission through donkey milk, together with the impact of different stages of infection on milk quality.