Background: Vulvovaginal candidiasis (VVC) is a significant public health concern characterized by increasing incidence and challenges in treatment. However, most studies investigating Candida spp. virulence factors and antifungal susceptibility predominantly rely on in vitro assays. While these assays are highly reproducible, they do not accurately replicate the complex vaginal microenvironment. To address this limitation, we developed an ex vivo model using porcine vaginal mucosa and a physiologically relevant volume of simulated vaginal fluid (SFV) to better mimic human vaginal conditions. Methods: Biofilm formation and fluconazole activity were assessed using the reference strain Candida albicans ATCC 90028 and two clinical isolates associated with VVC. Results were expressed as colony-forming units (CFU) and directly compared with in vitro assays conducted in Sabouraud dextrose broth (SDB) and SVF. Results: CFU analysis revealed that the ex vivo vaginal mucosa model supported more robust biofilm development, with counts ranging from 6.67 × 10⁷ to 7.20 × 10⁷ CFU/mL, compared to the in vitro SDB assay (3.58 × 10⁷ to 4.5 × 10⁷ CFU/mL). This suggests enhanced fungal growth under tissue-based conditions. Moreover, fluconazole achieved greater biofilm eradication in the ex vivo model (>70%) compared to the in vitro SDB assay (≤34.50%), which may indicate increased antifungal activity within a physiologically relevant environment. Conclusions: The ex vivo vaginal mucosa model offers a physiologically relevant platform for supporting C. albicans biofilm development and serves as a valuable alternative for preclinical screening of antifungal agents.