Calothamnus quadrifidus R.Br has many traditional uses and there are few reports about its chemical and biological activities. So our aim is to isolate the triterpenoidal compounds from dichloromethane fraction (DCMF) of Calothamnus quadrifidus R.Br leaves and in addition to evaluate the antibacterial activity of the isolated compounds. DCMF of C. quadrifidus leaves was subjected to different chromatographic techniques to isolate pure triterpenoidal compounds which were identified using different chemical and spectroscopic techniques. Antibacterial activities of the isolated compounds were evaluated using agar well diffusion method while minimum inhibitory concentration was assessed by microtiter plate assay method. Five compounds were isolated and they were betulinic acid (1), ursolic acid (2), 3-acetyl-23-hydroxy betulinic acid (3), 2,23-dihydroxy betulinic acid (4) and 2,21,23-trihydroxy betulinic acid (5) were isolated from DCMF of C. quadrifidus leaves for the first time. Compounds 4 and 5 showed strong antibacterial activity against S. typhimurium while compound 4, 5 and 3, 4 exhibits moderate effect against E.coli and S. aureus respectively. Pure triterpenoidal compounds isolated from C. quadrifidus leaves showed antibacterial activities in different strengths.