Cutaneous leishmaniasis (CL) is firmly established in South America. We aimed to assess the detection of IgG antibodies against 14 and/or 16 kDa antigens by immunoblot (IB) for CL serological diagnosis in French Guiana, an area where many endemic pathogens could interfere with it. This study was performed retrospectively on sera from 141 patients at the Cayenne tertiary hospital: 30 were patients with confirmed CL, 71 were diagnosed with various other endemic pathogens, 11 were diagnosed with an autoimmune disease, and 29 controls had no history of CL. Antibodies bound to the 14 and/or 16 kDa antigens in 27 of the 30 CL patients' sera and in 39 of the 111 non-CL patients' sera (26 from the infectious diseases group, four from the autoimmune diseases group, and nine from the dermatology department). The method tested showed a high sensitivity (90%) and a low specificity (66%), and a diagnosis odds ratio of 17.5 (95% CI [4.6-78.0]). This IB may be helpful to exclude the diagnosis of CL, prompting physicians to look for another diagnosis in the case of a negative IB.