Leishmaniases are an ensemble of diseases caused by the protozoan parasite of the genus Leishmania. Current antileishmanial treatments are limited and present main issues of toxicity and drug resistance emergence. Therefore, the generation of new inhibitors specifically directed against a leishmanial target is an attractive strategy to expand the chemotherapeutic arsenal. GDP-Mannose Pyrophosphorylase (GDP-MP) is a prominent therapeutic target involved in host-parasite recognition which has been described to be essential for parasite survival. In this work, we produced and purified GDP-MPs from L. mexicana (LmGDP-MP), L. donovani (LdGDP-MP), and human (hGDP-MP), and compared their enzymatic properties. From a rationale design of 100 potential inhibitors, four compounds were identified having a promising and specific inhibitory effect on parasite GDP-MP and antileishmanial activities, one of them exhibits a competitive inhibition on LdGDP-MP and belongs to the 2-substituted quinoline series.