The fatty acids (±)-2-methoxy-6Z-heptadecenoic acid (1), (±)-2-methoxy-6-heptadecynoic acid (2) and (±)-2-methoxyheptadecanoic acid (3) were synthesized and their inhibitory activity against the Leishmania DNA topoisomerase IB enzyme (LdTopIB) determined. Acids 1 and 2 were synthesized from 4-bromo-1-pentanol, the former in ten steps and in 7% overall yield, while the latter in seven steps and in 14% overall yield. Acid 3 was prepared in six steps and in 42% yield from 1-hexadecanol. Acids 1-3 inhibited the LdTopIB enzyme following the order 2 > 1 ⪢ 3, with 2 displaying an EC(50) = 16.6 ± 1.1 μM and 3 not inhibiting the enzyme. Acid 1 preferentially inhibited the LdTopIB enzyme over the human TopIB enzyme. Unsaturation seems to be a prerequisite for effective inhibition, rationalized in terms of weak intermolecular interactions between the active site of LdTopIB and either the double or triple bonds of the fatty acids. Toxicity towards Leishmania donovani promastigotes was also investigated resulting in the same order 2 > 1 > 3, with 2 displaying an EC(50) = 74.0 ± 17.1 μM. Our results indicate that α-methoxylation decreases the toxicity of C(17:1) fatty acids towards L. donovani promastigotes, but improves their selectivity index.