We evaluated the efficacy of enrofloxacin, alone or combined with metronidazole, against Leishmania infantum. The in vitro activity of this fluoroquinolone was assessed using two different methods: a direct test aimed at assessing the drug activity on the parasite, and an indirect test aimed at evaluating the drug effect on macrophage killing, lymphomonocyte activation and nitric oxide production. An in vivo test was also performed on 36 dogs with leishmaniasis, subdivided into three groups, one treated with enrofloxacin, another with enrofloxacin plus metronidazole, and a control group with meglumine antimoniate. The direct test did not show any action of enrofloxacin on the parasite, while the indirect testing showed an enhancement of macrophage killing and an increase in nitric oxide production. These findings show that enrofloxacin does not exert a direct anti-leishmanial activity in vitro. However, on the basis of the positive immunostimulation results shown in vitro and the clinical improvement, particularly of the cutaneous lesions, obtained in several dogs in the in vivo trial, the use of enrofloxacin in association with a specific anti-leishmanial drug can be proposed in the therapeutic protocol of canine leishmaniasis.