Cutaneous leishmaniasis is a widespread tropical disease caused by different species of Leishmania protozoa that are transmitted by infected sandflies. Clinical presentations are extremely diverse and dependent on a variety of parasite and host factors that are poorly understood. Leishmania (V.) braziliensis infection may result in a devastating disease manifestation characterized by the development of destructive lesions in the oral, nasal, and pharyngeal mucosal. Ecto-nucleotidases are enzymes that are involved in the hydrolysis of extracellular nucleotides. These enzymes have been shown to correlate with virulence of Leishmania parasites. In this work, we evaluated the ecto-nucleotidase activity of promastigotes from the twenty three different L. braziliensis isolates. We demonstrated that isolates obtained from mucosal lesions present higher levels of ecto-nucleotidase activity than those from cutaneous lesions. In addition, we show that in the murine model of cutaneous leishmaniasis, promastigote forms of parasite with higher activity induce a delayed/decreased immune response that may correlate with spreading of the parasites throughout the body. Thus, we propose that the level of ecto-nucleotidase activity of promastigotes may be a marker for the development of severe clinical forms of cutaneous leishmaniasis and also a possible target for future therapeutic intervention.