The hydroxyl group in ring A of pentacyclic triterpene is essential for antibacterial activity. Pentacyclic triterpenes bearing three hydroxyl groups in ring A were mainly found in plants and displayed significant antibacterial activity. However, no study reported how to obtain this type of compounds by chemical modification. In this study, twenty-five new pentacyclic triterpenes bearing polyhydroxyl ring A were synthesized from parental compounds ursolic acid (UA) and oleanolic acid (OA). Here, we showed that most of these derivatives displayed a significantly increased activity against Gram-positive bacteria compared to parental compounds in vitro. Some of these compounds exhibited minimum inhibitory concentration values of 1-3-fold more potent than the positive controls. The structure-activity relationship studies demonstrated that introducing two hydroxyl groups at positions C-1 and C-2 together with a small alkyl ester group at C-17 of UA and OA strongly enhanced growth-inhibiting activity against Gram-positive bacteria. The antibacterial mechanism of the active derivatives was shown to be involved in regulating the expression of genes associated with peptidoglycan and respiratory metabolisms, as well as virulence factor in bacteria. The enhanced potency and unique mechanism of action of these new pentacyclic triterpenes make them a promising antibacterial agent for further studies.