The microbicidal activity of a W/O/W multiple emulsion destined for vaginal application, containing lactic acid in the internal aqueous phase, octadecylamine (ODA) in the oily phase and benzalkonium chloride (CBZ) in the external aqueous phase was evaluated against three microbial strains: Escherichia coli, Staphylococcus aureus and Candida albicans. The results were different depending on the procedure used. Interpretable results were obtained if only a gentle agitation was used just after the introduction of the microbial suspension to the product. This suggested that vigorous agitation lead to a variable fraction of CBZ or ODA entrapped in the micelles of ethylene and propylene oxide copolymer (COE).