Background/Objective: American tegumentary leishmaniasis is a neglected tropical disease with limited therapeutic options characterized by high toxicity and poor tolerability. Drug repurpose offers a pragmatic strategy to accelerate the development of safer treatments. This study evaluated the antileishmanial activity of three clinically approved acetylcholinesterase (AChE) inhibitors—donepezil hydrochloride (DH), rivastigmine tartrate (RT), and galantamine hydrobromide (GH), tested individually and in combination with amphotericin B (AmpB) against Leishmania species relevant to tegumentary leishmaniasis. Methods: Antileishmanial activity was assessed against Leishmania (Leishmania) amazonensis promastigotes and intracellular amastigotes and Leishmania (Viannia) braziliensis promastigotes and axenic amastigotes. Cytotoxicity was evaluated in mammalian cell lines. The synergy with AmpB was analyzed at different proportions. Mechanistic studies included morphological analysis using light and scanning electron microscopy, flow cytometry, AChE activity assays, choline supplementation experiments, and membrane fluidity measurements. Results: All three AChE inhibitors demonstrated antileishmanial activity with selectivity indices > 1. DH emerged as the most promising candidate (IC₅₀ = 16.82 μM against promastigotes; SI = 10.25), with superior potency compared to other repurposed drugs. Strong synergistic interactions with AmpB were observed for all inhibitors (χΣFIC ≤ 0.17), with DH-AmpB displaying the most robust synergy (χΣFIC = 0.09), reducing the IC ₅₀ of AmpB by nearly 90-fold. DH induced distinct morphological alterations and acted through non-cholinergic mechanisms. The DH-AmpB combination retained maximal efficacy against L. (V.) braziliensis, with enhanced activity against clinically relevant amastigotes. Conclusions: Repurposed AChE inhibitors, particularly donepezil hydrochloride, are highly promising therapeutic candidates for tegumentary leishmaniasis. The robust synergistic effect with amphotericin B, together with their favorable safety profiles and non-antimicrobial mechanisms, positions these drugs as viable partners in dose-sparing combination regimens that could improve treatment adherence and reduce toxicity in endemic areas.

American tegumentary leishmaniasis (ATL) remains a therapeutic challenge due to the toxicity and limited effectiveness of conventional agents. This study evaluated Enterococcus mundtii CRL35, a food-grade lactic acid bacterium, as a dual-mechanism therapeutic platform against Leishmania (L.) amazonensis. Initial screening identified E. mundtii CRL35 as the most active strain among four enterococci tested, demonstrating high selectivity against promastigotes (SI = 95) and intracellular amastigotes (SI = 25). In a BALB/c murine model of cutaneous leishmaniasis, three treatment modalities were evaluated: topical application of cell-free supernatant (CFS-T), oral viable bacteria (LB-O), and their combination (CFS-T+LB-O). All three treatments achieved significant reductions in lesion parasite burden (approximately 2–3 orders of magnitude versus untreated controls), substantially exceeding the parasitological efficacy of amphotericin B under identical experimental conditions. CFS-T and CFS-T+LB-O additionally produced significant macroscopic lesion improvement. Importantly, no treatment was associated with adverse changes in organ indices or serum biochemical parameters. E. mundtii CRL35-based treatments promoted higher IgG2a/IgG1 antibody ratios compared to untreated controls and amphotericin B, generating an immune profile more favorable for intracellular parasite control. Taken together, these findings support the therapeutic potential of these modalities as safe, effective, and affordable alternatives for ATL, particularly in resource-limited endemic regions where toxicity and logistical barriers limit access to conventional therapy.