Background/Objectives: Cutaneous leishmaniasis (CL) remains a global health challenge, with treatment options often limited by drug resistance and systemic toxicity. Amphotericin B (AmB) represents a promising alternative. but intravenous administration causes severe systemic adverse effects. Despite growing interest in topical therapies, knowledge gaps remain regarding the comparative efficacy of delivery systems, including the influence of treatment timing and potential intrinsic effects. This study aimed to develop and characterize different topical AmB formulations (polymeric nanoparticles (PCL-AmB), a lipid-based (Oil_AmB) formulation, and a gel emulsion) to evaluate their in vivo efficacy against CL in a murine model, considering treatment initiation timing and potential intrinsic effects of the delivery systems. Methods: Formulations were prepared and characterized in terms of hydrodynamic size, polydispersity index, and AmB content. Antileishmanial activity was assessed in two independent in vivo experiments, with topical monotherapy administered five days per week for four weeks, starting either 10 or 30 days post-infection, representing early and established chronic stages of infection, respectively. Results: All formulations exhibited nanoscale dimensions and high homogeneity, with the lipid system demonstrating superior AmB solubilization. Both PCL-AmB and Oil_AmB reduced parasite load in the footpad, with Oil_AmB also reducing parasite load in draining lymph nodes. Conclusions: PCL-AmB and Oil_AmB reduced lesions and parasite burden in L. amazonensis-infected mice. Treatment timing was critical, with early Oil_AmB also reducing parasite loads in draining lymph nodes. These findings suggest that topical AmB formulations may provide a promising alternative for CL treatment, though further studies are required to optimize efficacy and administration schedules.