Trichomonas vaginalis causes trichomoniasis, the most recurrent sexually transmitted infection (STI) worldwide. Genital mycoplasmas, not considered STI agents, are frequently isolated from the female genital tract. A symbiosis between Mycoplasma species and T. vaginalis has been described. The aim of this study was to conduct molecular-based analyses of vaginal specimens, thus assessing the prevalence of non-STI Mycoplasma infections. In total, 582 samples from female patients and an additional 20 T. vaginalis isolates were analyzed by PCR using Mycoplasma specific 16S rRNA primers, and the obtained PCR products were sequenced. Mycoplasma species were detected in 28.2% of the collected vaginal samples. Mycoplasma hominis was found in 21.5% of the specimens, Ureaplasma species were found in 7.5% of the samples. The molecular data of the newly described species, CandidatusMycoplasma girerdii, were obtained for the first time in Austria, in a sample also positive for T. vaginalis. Analyses of the cultivated T. vaginalis strains confirmed the presence of M. hominis in two out of 20 samples. A comparably high prevalence of genital mycoplasmas was revealed through advanced diagnostic assays, with M. hominis and U. parvum being the most prevalent species. The previously described symbiotic relationship between M. hominis and T. vaginalis was confirmed.