Regardless of intense research on intramammary infection (IMI), IMI or mastitis continues to be the most important and costly disease of dairy cattle. For several years, research has been focusing on mastitis caused by gram-positive and gram-negative organisms. In the past, mastitis caused by Prototheca spp. was considered rare and frequently associated with poor environmental conditions or insufficient milking hygiene. Prototheca spp. is known today as an emergent pathogen that can cause both clinical and subclinical cases of mastitis that are usually chronic and progressive. Importantly, there is no treatment available, and all positive cows should be removed from the herd. Prototheca spp. also have zoonotic potential, which represents a human health risk due to the increase consumption of contaminated raw milk. The aim of the study was to investigate the presence of Prototheca spp. originated from Ohio bulk tank milk (BTM) samples. In total, 384 BTM samples were cultured to investigate the presence of Prototheca spp. in dairy farms. To enhance sensitivity, selective media Prototheca isolation medium (PIM) and increase volume (0.1 mL of milk instead of 0.01 mL) was used. Plates were incubated at 37°C and checked every 24 h for 72 h. Prototheca spp. was then presumptively identified by colony morphology, specific staining (Methylene Blue) and endospore visualization. Based on the results from this study, prevalence (number of positive samples over population size) of Prototheca spp. was estimated to be 6% (22/384) in BTM from Ohio dairies. Prototheca spp. possess pathogenic potential for both humans and animals. Thus, it is crucial to investigate and describe its prevalence in bulk tank milk to develop improved prevention methods and control practices, to prevent further spreading and to protect both animal and human health.