Two new rifabutin analogs, RFA-1 and RFA-2, show high in vitro antimycobacterial activities against Mycobacterium tuberculosis. MIC values of RFA-1 and RFA-2 were <0.02 g/ml against rifamycin-susceptible strains and 0.5 g/ml against a wide selection of multidrug-resistant strains, compared to >50 g/ml for rifampin and 10 g/ml for rifabutin. Molecular dynamic studies indicate that the compounds may exert tighter binding to mutants of RNA polymerase that have adapted to the rifamycins.