Composite milk samples from 548 cows, and samples from feces, feed, bedding, water, liners (before and after milking), and the postdipping product were aseptically collected from 2 Italian dairy herds from February to November of 2006. Prototheca zopfii was isolated from 11.9% of milk samples, 15% of feces, and 33.3% of bedding samples. No viable cells of P. zopfii were observed in water before washing procedures, whereas 25 to 28.6% of samples from water used for washing both refrigeration tanks and milking equipment were contaminated with this yeast-like microalga. Analogously, the presence of P. zopfii was detected only on swabs collected from the liners after milking. Interestingly, in 1 of the 2 herds, water from the drinking trough was contaminated by viable cells of both P. zopfii and the related environmental species Prototheca stagnora. No viable cells were observed in cow feed. On the basis of the results presented herein, P. zopfii seemed to be widespread throughout the environments of dairy herds where outbreaks of bovine mastitis had occurred.