Objective: In view of the national key project for infectious diseases “ the retreatment research of tuberculosis”, we determined the in vitro anti-mycobacterial activity of four drugs in the treatment, pasiniazid (Pa), moxifloxacin (Mfx), Rifabutin (Rfb) and rifapentini (Rft) in combination to multidrug-resistant and extensively drug-resistant mycobacterium tuberculosis. Method: Three-dimensional checkerboard in Middlebrook 7H9 broth microdilutions was used to detect the fractional inhibitory concentration index (FICI) of anti-tuberculosis drug combination (MfxPa, MfxPaRfb and MfxPaRft) to twenty clinical isolates of Mycobacterium tuberculosis, including ten multidrug-resistant isolates and ten extensively drug-resistant isolates. The test results were interpreted by calculating the FICI to judge the in vitro synergy, with FICI < 0. 5 and FICI < 0.75 as the basis of two drugs and three drugs have synergy. Results: The FICI range of MfxPa combination for multidrug-resistant isolates and ten extensively drug-resistant isolates was 0.28-1, only three isolates <0.5, showed synergistic effect. The FICI range of MfxPaRfb combination for ten isolates of multidrug-resistant isolates was from 0.31 to 1.25, two isolates <0.75, showed synergistic effect and for ten isolates of extensively drug-resistant isolates was from 0.53to 1.25, five isolates <0.75, showed synergistic effect. The FICI range of MfxPaRft combination for ten isolates of multidrug-resistant isolates was from 0.16to 0.655, all showed synergistic effect and for ten isolates of extensively drug-resistant isolates was from 0.34 to 1.68, five isolates<0.75, showed synergistic effect. Conclusion: The synergism of MfxPa combination was poor. When a third agent (Rfb or Rft) was added to the combination, the synergistic effect was better. The MfxPaRft combination showed better synergism than MfxPaRfb combination.