AimsThis study aimed to investigate the application of Origanum vulgare L. essential oil (EO) as an alternative antimicrobial agent against vaginal infections. Methods and ResultsMicrodilution methods were applied for the detection of the minimal inhibitory (MIC) and the minimal bactericidal/fungicidal (MBC/MFC) concentration of 15 clinical strains originating from women with symptoms of vaginal infection. Optical density determination (OD) was used for detection of the Candida growth rate. Staining with DAPI was used to detect the influence of EO on nuclear condensation and fragmentation, while the brine shrimp bioassay was used to determine the toxicity of EO. Chemical composition analysis was done using GS-MS. According to the MIC and MBC/MFC values, the most susceptible strains to EO were: Escherichia coli 1, E.coli 2, Staphylococcus aureus 3 and Candida albicans 1-3. Inhibition of C. albicans filamentation was detected at 045lml(-1). ConclusionsThe obtained inhibition (%) from Candida growth curves points to a shorter period of time (24h) for determining IC50 as MIC and IC99 as MFC value. These values could be recommended as valid parameters for the faster detection of the effectiveness of EO on Candida isolates. Significance and Impact of the StudyExamination of potential of the O. vulgare EO as a main antimicrobial constituent within vaginalettes in gynaecological practice.