Dequalinium chloride (CAS 522-51-0) and povidone iodine (CAS 25655-41-8) are known as antiseptic agents and used in the local treatment of vaginal infections. Clotrimazole (CAS 23593-75-1) is an anti-fungal drug and applied primarily in the therapy of vulvo-vaginal candidiasis and to a lesser extent in bacterial vaginosis and trichomoniasis. However, antimicrobial activities of those three agents in comparison to each other have not been reported so far. To address this issue the antimicrobial activities of these agents against 18 germs relevant to vaginal infections were determined. The tested species are representatives of the genera Staphylococcus, Streptococcus, Enterococcus, Listeria, Escherichia, Proteus, Gardnerella, Bacteroides, Prevotella, Porphyromonas, Candida, and Trichomonas. All micro-organisms were susceptible to dequalinium chloride with the exception of Proteus mirabilis. At a given dose, the activity of dequalinium chloride was higher as compared to the other substances. In view of its wide antimicrobial spectrum dequalinium chloride is an efficient alternative in the local therapy of vaginal infections such as fluor vaginalis, bacterial vaginosis, aerobic vaginitis, vulvo-vaginal candidiasis and trichomoniasis.