OBJECTIVE To determine the prevalence of Gardnerella vaginalis, anaerobic bacteria and Mycoplasma hominis in vaginal specimens of women with and without bacterial vaginosis (BV) as well as to determine the sensitivity and specificity of the direct sialidase assay of vaginal fluid as a rapid test for diagnosing this syndrome. METHODS Vaginal cultures were obtained from 109 nonpregnant women (mean age 33 +/- 7.1 years), 47 of them with clinical signs of BV (BV+) and 62 of them without BV (BV-). In addition, we determined the vaginal sialidase activity in both groups, which may serve as a feature of this syndrome. RESULTS Anaerobic bacteria were isolated in 91% and 18% of the BV+ and BV- groups, respectively (p < 0.001). Peptostreptococcus spp., Prevotella bivia and Porphyromonas spp. were strongly associated with BV. P. bivia and Prevotella spp. represented 44% of all the anaerobes isolated in the BV+ group. All the isolated P. bivia strains presented sialidase activity. G. vaginalis and M. hominis were isolated in 76% and 42% of the BV+ and 1% and 0% of the BV- women, respectively (p < 0.001). Mobiluncus morphotypes were observed in 34% of the BV+ and 0% of BV- women. Sensitivity, specificity, positive predictive value and negative predictive value of sialidase activity were 81%, 94%, 90% and 86%, respectively. CONCLUSIONS Our data demonstrate a strong association between G. vaginalis, M. hominis, and P. bivia and BV. Sialidase activity and Gram stain of vaginal fluid represent accurate methods for diagnosing BV.