Vulvovaginal candidiasis is a common fungal infection that affects the female genital system. Resistance to antifungals is an emergent problem worldwide. We aimed to detect the prevalence of different Candida species associated with vulvovaginal candidiasis, determine their antifungal susceptibility pattern, and evaluate the molecular mechanisms associated with Fluconazole resistance. Methodology: This study included 300 patients. Candida species have been identified phenotypically. Antifungal susceptibility was tested using a disc diffusion method. The molecular mechanisms of Fluconazole-resistance were determined by analyzing the expression levels of Fluconazole target, efflux pump and efflux pump-regulator genes by RT-PCR. Results: Candida spp were detected in 75/300 (25%) of cases. The most frequently isolated species was C. albicans (61.8%), whereas the predominant species of non-albicans was C. glabrata (29%). Nystatin was the most effective agent. Fluconazole-resistance was observed markedly in C. glabrata (54.5%), and efflux-pump was the predominant mechanism of resistance, which was associated with overexpressed CgPDR1, CgCDR1 and CgSNQ2 genes. Upregulation of the efflux-pump genes and their regulator were associated with cross-resistance to different azoles. Conclusion: C. glabrata is a common cause of non-albicans vulvovaginal candidiasis. The majority of clinical resistance in C. glabrata is attributed to the upregulation of efflux-pump genes.