The rise in azole resistance among Nakaseomyces glabratus and Pichia kudriavzevii in recurrent vulvovaginal candidiasis presents a growing public health challenge. This study investigated the expression of antifungal resistance-related genes (ERG11, CDR1, CDR2, and MDR1) in clinical resistant (CR) and clinical and laboratory resistant (CLR) strains of these yeasts. Cervicovaginal samples from patients with recurrent infections were collected, microscopically examined, and cultured. Yeast species were identified phenotypically and genotypically, followed by drug sensitivity testing. Total RNA was extracted, reverse transcribed to complementary DNA, and real-time polymerase chain reaction was used to quantify target gene expression, comparing results to drug-sensitive controls. Non-Candida albicans species constituted 29% (45 cases) of the isolates, with N. glabratus (68%) and P. kudriavzevii (17%) being the dominant species. Other species included Candida parapsilosis, Meyerozyma guilliermondii, Candida orthopsilosis, Saccharomyces cerevisiae, and Rhodotorula mucilaginosa. Coinfections with P. kudriavzevii/C. albicans and N. glabratus/C. albicans were also observed. Ketoconazole, itraconazole, and 5-flucytosine demonstrated the best antifungal activity against most species. However, some N. glabratus isolates were resistant to miconazole, clotrimazole, and amphotericin B, while all P. kudriavzevii isolates resisted clotrimazole. Overexpression of the CDR1 gene was noted in N. glabratus (CR, 21.53 ± 1.26; CLR, 84.96 ± 0.67), and the ERG11 and CDR1 genes in P. kudriavzevii (ERG11 for CR, 28.56 ± 2.16; CDR1 for CLR, 35.89 ± 0.35). These results indicate that even in cases where an isolate is classified as susceptible by drug susceptibility testing, elevated gene expression may persist, and treatment should not be discontinued.