Cutaneous leishmaniosis (CL) is considered as one of the most important tropical diseases. Herbal therapy is the ideal treatment for CL because of the reduced injection pain, availability, lower cost and non-toxicity effects. The present study aimed to evaluate the in vivo antileishmanial activity of concocted herbal topical preparation (Aloe vera, Perovskia abrotanoides, Nigella sativa, propolis, lavender and olive oil) to evaluate its efficacy against Leishmania major (MRHO/IR/75/ER) in comparison to the gold standard treatment. Following the cause of cutaneous leishmaniosis, the BALB/c mice were divided into three groups, test group (ointment formulation), positive control (Glucantime) and negative control (untreated), respectively, which were treated twice a day for 28 consecutive days. The lesion size and parasite burden were evaluated for in vivo evaluation. The herbal topical ointment was able to significantly decline the lesion progression and reduce parasite burden in mice inoculated with L. major promastigotes in the test group compared with the negative control group (P=<0.001). In mice treated with the formulation, the number of amastigotes significantly decreased (P=<0.001), compared with that in the negative control group. Moreover, comparative features of both treatments showed there was no difference between the herbal-treated and glucantime-treat mice (P=0.63). The herbal topical ointment displayed significant in vivo antileishmanial activities. It may be that using ointment formulation beside other skin repair compounds can be used as an alternative medicine in the treatment and healing of human CL lesions. Further investigations are needed to study the pharmacologic and pharmacokinetics aspects of ointment formulation in the treatment and healing of human CL lesions.