Objective To validate the use of Seegene Allplex™ Vaginitis assay in the diagnosis of candidiasis, bacterial vaginosis (BV) and trichomoniasis. Design Cross‐sectional, prospective study conducted in a single centre. Setting Outpatient clinic of a gynaecology department. Population Consecutive symptomatic and asymptomatic women (18–60 years of age). Methods Comparison of the assay test with the reference standards for the diagnosis of vaginitis (cultures for yeasts, Nugent for BV and nucleic acid amplification test for trichomoniasis). Main outcome measures Performance of the investigational assay, in comparison with the reference standards for the diagnosis of the presence of Candida spp., Trichomonas vaginalis and BV. Secondary objectives are the evaluation of the performance of the test in postmenopausal women and in symptomatic women. Results A diagnosis of vaginitis was established in 14.0%. The global prevalences of BV, Candida spp. and T. vaginalis were 22.3%, 13.2% and 2.4%, respectively. The sensitivity and specificity of the assay test for those three causes of vaginitis were as follows: BV 91.7% and 86.6%; any Candida spp. 91.1% and 95.6%; Candida albicans 88.1% and 98.2%, non‐albicans Candida 100% and 97.5%, and T. vaginalis 94.4 and 99.9%. The performance of the test was identical in the subgroup of women that reported vulvovaginal symptoms. The presence of multiple infections did not interfere with the performance of the test. Conclusions The Seegene Allplex™ Vaginitis assay has an excellent performance in the diagnosis of the BV and presence of Candida; the results were good for trichomoniasis, but the study was underpowered for this outcome. Tweetable abstract Seegene Allplex™ Vaginitis is an excellent option for screening and diagnosis of vaginitis.