A poloxamer surfactant, CRL8131, was evaluated for activity against Mycobacterium tuberculosis (Erdman) by itself and in combination with antibiotics in broth culture, in a macrophage cell line assay, and in testing with mice. In the broth culture, CRL8131 suppressed the growth of M. tuberculosis and produced synergistic effects in combination with isoniazid, rifampin, and streptomycin. It also displayed synergy with isoniazid and rifampin against two drug-resistant isolates. In the macrophage cell line assay, CRL8131 produced a synergistic effect on intracellular killing of M. tuberculosis by isoniazid, rifampin, streptomycin, pyrazinamide, thiacetazone, D-cycloserine, ethionamide, amikacin, clindamycin, and p-aminosalicylic acid. It demonstrated no synergy or antagonism with ethambutol, gentamicin, kanamycin, ciprofloxacin, or nalidixic acid. Finally, with C57BL/6 mice infected with M. tuberculosis, a combination of CRL8131 and either thiacetazone or pyrazinamide produced 100% survival at 40 days whereas the antibiotics produced only 33% survival and CRL8131 produced 0% survival when used as single agents. This improved survival rate was associated with a significant reduction in the number of organisms in the lungs and spleens of infected mice.