ATB-152E and ATB-152J are structurally similar anti-tuberculosis (anti-TB) chemicals developed by our group in previous research. To study their mechanisms, Mycobacterium tuberculosis (M. tuberculosis) H37Ra strains spontaneously resistant to them were collected and subjected to colony morphology analysis and biofilm formation assay. In total, seventeen ATB-152E resistant strains and fifteen ATB-152J resistant strains were studied. The frequencies of resistance to both compounds were 10^－7. Compared with the wild type strains of H37Ra, ATB-152E resistant strains had more folds, while ATB-152J resistant strains presented a flatter pattern with reduced folds. Meanwhile, the time of biofilm formation of resistant strains also differed from the wild type strains, indicating a possible mechanism of inhibition via lipid metabolism. The acquisition of the strains resistant to ATB-152E and ATB-152J could lay a foundation to further study of them.