Dientamoeba fragilis is a commonly encountered trichomonad which has been implicated as a cause of gastrointestinal disease in humans. Despite the frequency of reports recording infections with this parasite, little research has been undertaken in terms of antimicrobial susceptibility. The aim of this study was to evaluate the susceptibility of D. fragilis to several commonly used antiparasitic agents: diloxanide furoate, furazolidone, iodoquinol, metronidazole, nitazoxanide, ornidazole, paromomycin, secnidazole, ronidazole, tetracycline, and tinidazole. Antibiotic susceptibility testing was performed on four clinical strains of D. fragilis, designated A, E, M, and V, respectively. Molecular testing followed, and all strains were determined to be genotype 1. The activities of antiprotozoal compounds at concentrations ranging from 2!g/ml to 500!g/ml were determined via cell counts of D. fragilis trophozoites grown in dixenic culture. Minimum lethal concentrations (MLCs) were as follows: ornidazole, 8 to 16 !g/ml; ronidazole, 8 to 16!g/ml; tinidazole, 31!g/ml; metronidazole, 31!g/ml; secnidazole, 31 to 63!g/ml; nitazoxanide, 63 !g/ml; tetracycline, 250!g/ml; furazolidone, 250 to 500!g/ml; iodoquinol, 500!g/ml; paromomycin, 500!g/ml; and diloxanide furoate,>500!g/ml. This is the first study to report the profiles of susceptibility to a wide range of commonly used treatments for clinical isolates of D. fragilis. Our study indicated 5-nitroimidazole derivatives to be the most active compounds in vitro against D. fragilis.