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A series of piperazine derivatives containing bis‐isoxazole were successfully synthesized by the reaction between piperazine and substituted isoxazole derivatives under the optimal reaction condition. The structures of all products were confirmed by IR, HRMS, 1H and 13C NMR. The antibacterial activities of all products against Mycobacteria smegmatis in vitro were tested using rifampin as a positive control. Two compounds presented wonderful anti‐mycobacterium activity, and their minimum inhibitory concentrations were respectively 2.60±1.11 μmol/mL and 4.35±0.00 μmol/mL, which indicated that they can be regarded as candidates for development of anti‐TB drugs. [ABSTRACT FROM AUTHOR]