The prevalence of bovine Tritrichomonas foetus infection has decreased almost to zero in most European countries, such as Poland, which has been Tritrichomonas foetus-free since 1997. However, trichomonosis is a notifiable disease and there is a duty to examine samples from cattle. In this study, we present an unexpected cross-reaction with Honigbergiella-like DNA in a specimen from a bull. The bovine sample was submitted to the Department of Parasitology National Veterinary Research Institute in Pulawy (NVRI) for confirmatory testing after having been examined at the Regional Veterinary Laboratory, during a routine T. foetus diagnosis. Positive results from microscopic observation and cultures were confirmed. Noteworthily, parasites grew on Diamond’s medium only after seven days of incubation, while optimal growth of trichomonads is generally observed after two to four days for this medium. Moreover, by using PCR we obtained positive results for the presence of T. foetus. However, sequencing of the amplification product revealed 99.62% identity with Honigbergiella sp. Our data suggest that false-positive results may occur in commonly used PCR tests. Thus, unexpected results should be carefully interpreted