Background <p><i>Candida</i> spp. are microorganisms present in the microbiota of healthy adults. An imbalance between the microbiota and the host can cause oral candidiasis. <i>Candida</i> spp. infection has been linked to altered salivary gland function and specific oral mucosa lesions. However, there is limited research on the prevalence of <i>Candida</i> spp. in different oral mucosal lesions. This study aimed to evaluate the prevalence of <i>Candida</i> spp. across various oral mucosa lesions, regardless of symptom presence, and to contribute to delineating the profile of colonization/infection, by identifying predisposing local and systemic risk factors, performing molecular identification of <i>Candida</i> species, and evaluating the antifungal susceptibility of the isolated yeasts.</p> Methods <p>A cross-sectional study was conducted, involving the collection of oral mucosal samples with sterile swabs from two sites: the lesion and the control site. Patients were excluded if they received antifungal, corticosteroid, or local antibiotic therapy within 15 days, or systemic antibiotics within 30 days before consultation. The samples were processed using CHROMagar <i>Candida</i>, followed by identification through biochemical and morphological tests. The MALDI-TOF MS methodology confirmed the identification of all isolates. The isolates were then evaluated for in vitro antifungal susceptibility. In addition, general and dental health data were collected to identify predisposing factors. Pearson’s chi-square test, McNemar’s test, Fisher’s exact test, or Student’s t-test was applied. A significance level of <i>p</i> &lt; 0.05 was used.</p> Results <p>40% of the 80 patients tested positive for <i>Candida</i> spp. within the oral mucosal lesions, with <i>C. albicans</i> (62.5%) being the most common isolated species. Diabetes was significantly associated with <i>Candida</i> spp. colonization (<i>p</i> = 0.01), and elevated nystatin minimum inhibitory concentrations (MICs) were observed in 37 isolates. It was further observed that oral lesions associated with risk factors influence <i>Candida</i> spp. colonization.</p> Conclusion <p>A high prevalence of <i>Candida</i> spp. was observed in oral mucosal lesions, particularly among patients with diabetes mellitus. In the absence of established clinical breakpoints or epidemiological cutoff values (ECOFFs) for nystatin, elevated MICs values may suggest reduced susceptibility among certain isolates; however, such interpretations should be made with caution. Although these results do not indicate resistance, they serve as a caution to clinicians regarding the potential for decreased antifungal effectiveness.</p> Clinical trial <p>Not applicable.</p>