Protothecosis is a potential zoonotic disease associated with bovine mastitis which can be transmitted to humans through contaminated milk. Considering the increasing prevalence of bovine mastitis due to Prototheca species, individual cow milk samples were analyzed using microbiological examination and biomolecular assay. Aspects related to health requirements for milk production, clinical and histological bovine mastitis were also described. The results showed 24/257 (9.3%) culture-positive samples and 42/257 (16.3%) PCR-positive samples. Moreover in 5 cows with somatic cell count over 106/mL presented histological features of mastitis. This study reveals that the presence of Prototheca species in dairy herds was related to the hygienic conditions of the milking equipment, showing an emerging public health issue.