BACKGROUND: Candida spp and Malassezia spp cause superficial infections that may be resistant to conventional treatments. Violet light-emitting diodes (LEDs) therapy is a therapeutic alternative. PURPOSE: To describe the protocol for evaluating the antifungal effect of violet LEDs 410 nm ± 10 nm on Candida spp and Malassezia spp in vitro. PROTOCOL: LEDs 410 nm ± 10 nm are applied to a fungal suspension at fluences of 61.13 J/cm2, 91.70 J/cm2, and 183.39 J/cm2. The isolates are cultured for 48 to 72 hours. Colony forming units (CFUs) are quantified by visual counting and percent culture plate occupancy by digital analysis. Morphology is assessed by light microscopy and Gram staining, and yeast metabolism/function by transmission electron microscopy, assessment of reactive oxygen species, and DNA fragmentation. DATA ANALYSIS: the percentage of LEDs inhibition is calculated considering the growth of the negative control condition and the percentage of plate occupancy by yeasts by dividing the number of pixels classified as colonies by the total number of pixels on the plate. The morphological and functional aspects are described for the intervention and negative control. The ANOVA test is used to compare the mean percentages of growth inhibition and plate occupancy between the three fluences of LEDs 410 nm ± 10 nm and the negative control. ESTIMATED RESULTS: We intend to determine the antifungal effect of the different fluences of LEDs 410 nm ± 10 nm on Candida spp and Malassezia spp. The evaluation of other fungal species by this protocol should be investigated.