The balance of the vaginal ecosystem results from synergistic interactions established between the different colonizing microorganisms. Lactobacillus spp. stands out as a predominant member, acting a crucial protective role in maintaining vaginal health. As vulvovaginal candidiasis remains prevalent and antifungal resistance grows, there is a pressing need for alternative therapies. However, any development of new therapies must carefully consider their impact on naturally colonizing species, particularly Lactobacillus spp., believed to be crucial in preserving vaginal homeostasis. This underscores the importance of assessing the protective effect of the oil on Lactobacillus spp. when exploring alternative therapies. This study evaluates the effect of the vapor phase of white thyme essential oil (VP-WTEO), on Candida albicans biofilm formation and on co-colonization of a reconstituted human vaginal epithelium (RHVE), with a specific emphasis on its effect on Lactobacillus gasseri. The VP-WTEO effect on both species was evaluated by DNA quantification and by microscopy, while the effect on the RHVE was assessed by the determination of lactate dehydrogenase activity. The results revealed that the VP-WTEO had a significant inhibitory effect on C. albicans biofilm formation and induced a significant reduction in their mature biofilms. Furthermore, it was observed a significant reduction in the number of C. albicans cells colonizing the RHVE without any alteration on the number of cells of L. gasseri in the presence of the VP-WTEO. This study suggests that the VP-WTEO could be a promising solution to treat and prevent VVC, being a safe alternative for the remaining vaginal microbiota.