BACKGROUND Vaginal infections lie among the most common causes women ask for medical advice. In order of frequency bacterial vaginosis, vulvovaginal candidiasis, and trichomoniasis are responsible for 90% of vaginitis/vaginosis. OBJECTIVE To evaluate a DNA hybridization test for simultaneous molecular detection of Gardnerella vaginalis, Candida species and Trichomonas vaginalis, as an alternative to conventional microbiological methods. METHODS Cohort, cross-sectional, and comparative study of 1,003 vaginal samples from symptomatic women from our health-care area. Two swabs were obtained from each woman, one for routine microbiological diagnosis of vaginal infection (wet mount, Gram stain, and mycological culture) and the other for the DNA hybridization test (Affirm VPIII, Becton Dickinson). This method detects clinically significant levels of G. vaginalis (2 x 10(5) CFU/ml), Candida spp. (1 x 10(4) cells) and T. vaginalis (5x103 trichomonads). RESULTS Out of the 1,003 women studied, 30.6% tested positive for bacterial vaginosis, 23.3% for vulvovaginal candidiasis, and 0.5% for trichomoniasis. The Affirm VPIII method turned out positive in 27.5%, 27.4% and 0.5% of cases, respectively. No statistically significant differences were found between the molecular technique and conventional methods for microbiological diagnosis of vaginitis/ vaginosis (p < 0.05). CONCLUSIONS The Affirm VPIII test correlated well with wet mount, Gram stain and mycological culture. Although its cost is relatively high, it is fast, reproducible, easy, and can be done in either clinical laboratories or Gynecology offices, which permits prescribing a specific early treatment.