One of the most important steps in preclinical drug discovery is to demonstrate the in vivo efficacy of potential leishmanicidal compounds and good characteristics at the level of parasite killing prior to initiating human clinical trials. This paper describes the use of dehydrothyrsiferol (DT), isolated from the red alga Laurencia viridis, in a pharmaceutical form supported on Sepigel, and the in vivo efficacy against a mouse model of cutaneous leishmaniasis. Studying the ultrastructural effect of DT was also carried out to verify the suspected damage at the cellular level and determine the severity of damages produced in the homeostasis of promastigotes. BALB/c mice infected with Leishmania amazonensis were divided into four groups: untreated mice, mice treated with miltefosine orally and mice treated topically with 1% and 0.5% DT-Sepigel; treatment was carried out for two weeks. Treatment with DT significantly reduced the parasite load in skin, liver and spleen compared with the untreated group. In addition, DT-Sepigel at the lowest concentration (0.5%) showed the best results, reducing lesion size by 87% at 3 weeks post-treatment. DT-Sepigel has demonstrated to be a potent topical treatment that, in combined drug trials, may aim at combating cutaneous leishmaniasis.