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The parabasalid Pentatrichomonas hominis is generally considered to represent a symbiotic component of the gastrointestinal microbiota in a wide variety of vertebrate hosts including humans. Nevertheless, a limited number of studies have raised the possibility that it may act as a pathogen of humans, dogs, and pigs and that some human infections may have a zoonotic origin. Data from North America revealed an association between P. hominis and the bovine urogenital tract, principally in bulls and rarely in cows. The importance of this observation is linked to possible interference in the accurate diagnosis of the economically important venereal pathogen Tritrichomonas foetus. The current study employed culture-based and molecular methods to examine the preputial cavities of four breeding bulls, raised in open pasture in southeastern Brazil, for the presence of trichomonads. Motile protozoa were isolated from three of the bulls and were definitively identified as P. hominis based on nucleotide sequencing of polymerase chain reaction (PCR) amplicons derived from the ribosomal RNA operon (ITS1-5.8S rDNA-ITS2) of the parasite. The potential implications of these findings for bovine and human health are discussed.