Studies on in vitro skin permeation and in vivo anti-leishmanial activity in mice experimentally infected with Leishmania (Leishmania) major pointed out to the potential of a new paromomycin (PA) formulation (hydrophilic gel) for treatment of cutaneous leishmaniasis (CL). In this study, the activity of this formulation was evaluated in animals experimentally infected by Leishmania species that prevail in the New World. PA gel activity was compared to antimony treatment, since it is still the first choice treatment to the different clinical forms of leishmaniasis. The topical treatment activity with 10% PA gel in BALB/c mice infected by Leishmania (Leishmania) amazonensis was higher than that observed for parenteral antimony treatment, while the efficacy of these two regimes in hamsters infected by Leishmania (Viannia) braziliensis was similar. These results suggest that this formulation could be suitable for clinical studies and may represent an alternative novel formulation for topical treatment of CL.