Background: Prototheca Spp. belongs to family Chlorellaceae and can cause mastitis in dairy cattle herds. Several Prototheca species could be isolated in different parts of the dairy herds. Objectives: The objectives were to determine: (1) the investigation status of bulk tank milk Prototheca Spp prevalence in industrial dairy cattle herds in different area of Iran. And (2). To evaluate the fungal agent prevalence of bulk tank milk of dairy cattle herds in different seasons. Materials and Methods: Bulk tank milk for the presence of Prototheca and fungal agents from 165 industrial dairy cattle herds complexes were tested. The population of each herd were about 850–3500 Holstein dairy cows. The samples were taken from 32 cities and 16 provinces of Iran, respectively. In total, almost 66800 Holstein dairy cows represented. The population of lactating cows included about 25800. Samples were cultured on sabouraud glucose agar (SGA) medium with chloramphenicol (100 mg / l). Results: Only 8 (4.84%) out of 165 samples were isolated for Prototheca Spp. It had been isolated in different seasons. They were 2, 4, 1 and 1 samples in spring, summer, autumn and winter, respectively. Candida, Fusarium and Trichosporon Spp. were isolated from 137, 2 and 1 samples, respectively. According to the obtained results Prototheca and fungal agents are present in bulk tank milk of industrial dairy cattle herds in different provinces of Iran. Conclusion: It is concluded that exposure to Prototheca and different fungal agents could be common in the dairy cattle herds in Iran. The results are important as dairy cattle health and human sanitation hazards.