We have demonstrated that fluoroquinolones, a class of antibacterial agents that act through inhibition of type II DNA topoisomerases, exert selective action against intracellular amastigotes of Leishmania (Viannia) panamensis at concentrations that are achievable in vivo. Drug cytotoxicity assays employing the luciferase reporter gene revealed that intracellular amastigotes were 6.6- to 25.9-fold more sensitive than human macrophages (P < 0.05) to second-generation fluoroquinolones in vitro. The most selective agents (enoxacin and ciprofloxacin) exhibited 2 orders of magnitude greater potency against parasites (50% effective dose [ED₅₀] = 54.9–83.4 μM) than host cells (ED₅₀ = 1,425–1,740 μM). Linear regression analysis of ED₅₀ data confirmed a complete lack of correlation (r = 0.001) between the relative drug sensitivities of parasites and host cells. A potential relationship between the structures of fluoroquinolones and their relative leishmanicidal activities was observed. The key substituents of the basic pyridone β-carboxylic acid nucleus accounting for enhanced antiparasite potency and selectivity appear to be a nitrogen at position 8 of the bicyclic nucleus (enoxacin), a cyclopropyl substituent at the R₁ site (ciprofloxacin), and linkage of the R₁ and X₈ groups by a CH₃CHO bridge to form a tricyclic compound (ofloxacin). These findings support the potential of fluoroquinolones and derivatives as novel antileishmanials and encourage their clinical evaluation.