Bixin is a lipophilic apocarotenoid abundant in the aril of L. seeds. Widely used as a natural colorant in food, textile, and cosmetic industries, its pharmacological potential remains underexplored. This study aimed to extract and purify bixin, characterize its physicochemical properties, develop and validate an HPLC method for quantification, and evaluate its antifungal activity, cytotoxicity, and selectivity . Extraction was performed by Soxhlet using hexane and chloroform, followed by recrystallization in acetone. Characterization (TG/DTA, FTIR, and H NMR) confirmed predominance of the 9'- isomer. The HPLC method (λ=470 nm) was validated per ICH Q2-(R2), showing specificity, linearity (R = 0.9993), low LOD/LOQ (0.638 and 1.934 μg mL), and high precision and accuracy (RSD < 2%; recovery 99.4-100.8%). Antifungal assays against eight strains revealed moderate activity, with MICs from 2 to 256 μg mL. The best results were observed for and (MIC = 2 and 4 μg mL). Bixin also inhibited virulence factors in Cytotoxicity on Vero cells showed a CC of 30.71 μg mL, with selectivity indices of 15.36 and 7.68 for and, respectively. These results support bixin's potential as a natural antifungal agent.