In our present study, the hexane fraction from the root of the Thai medicinal plant Strophioblachia fimbricalyx Boerl. was purified and the purification led to the isolation of 3-acetylaleuritolic acid, trigonostemone and 3,6,9-trimethoxyphenanthropolone. The aims of this work are to evaluate antibacterial activity of these three isolated compounds from our local plant and to study their mechanism of actions toward target pathogenic bacteria (Gram positive and Gram negative bacteria). The antibacterial activity of isolated compounds was primary screened by agar well diffusion method and the active compound was subjected to determine for MIC and MBC values by microdilution method. The kinetic study of the bacteriostatic or bactericidal activity time-kill experiment (24 h) and mechanism of action on cell morphology toward target bacteria detected by scanning electron microscope of the active compound were further evaluated. Results indicate that among the tested three compounds, trigonostemone was the only active one. It exhibited an inhibitory effect on the growth of Gram positive bacteria , methicillin-susceptible Staphylococcus aureus (MSSA) DMST 2933, methicillin-resistant S. aureus (MRSA) DMST 20651 and Bacillus cereus ATCC 11778 with the MIC/MBC values of 12.5/25.0, 6.25/6.25 and 6.25/6.25 mg/mL, respectively . Trigonostemone possessed time- and concentration-dependent bacteriostatic activity against S. aureus (MSSA) DMST 2933 and bactericidal activity against B. cereus ATCC 11778. It caused bacteriostatic activity against S. aureus (MSSA) DMST 2933 at the concentration of 2 × MIC by changing cell morphology and bactericidal activity against B. cereus ATCC 11778 at the concentration of 2 × MIC after 4 h by inducing cell size variations at the concentrations of 2 × MIC, respectively. This finding suggests that trigonostemone isolated from the root of S. fimbricalyx has a potential to be used as natural antibacterial compound against S. aureus (MSSA) DMST 2933 and B. cereus ATCC 11778 bacterial strains.