Luo Han Guo (LHG) fruits (Siraitia grosvenorii Swingle) have been used as traditional medicine in China for centuries to treat sore throats and coughs. However, LHG leaves are seldom used and minimal scientific information is available on them. In our recent study on the leaves of S. grosvenorii, the bioactive compounds β-amyrin (2), aloe emodin (5), aloe-emodin acetate (6), 5α,8α-epidioxy-24(R)-methylcholesta-6,22-dien-3β-ol (7) and p-hydroxyl benzyl acid (8), accompanied by n-hexadecaoic acid (1), 12-methyltetradecanoic acid (3), β-sitosterol (4) and daucosterol (9) were first obtained. Their structures have been identified on the basis of spectroscopic studies. The activities of these compounds were evaluated in vitro against the growth of oral bacterial species Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum and the yeast Candida albicans, and their minimum inhibition concentrations were determined. Furthermore, the activity against S. mutans is likely to be due to the inhibition of glucosyltransferase. The experimental data provide important information on bioactive phytochemicals in the leaf of S. grosvenorii, which shows that the leaf can be a new resource as an antibacterial agent.