Ginsenosides are the major active constituents in both white and red American ginseng (AG), and their pharmacological effects on central nervous, cardiovascular, and endocrine systems have been well elucidated. However, the anti-Propionibacterium acnes (P. acnes) activity of them is still unknown. In this study, 5 ginsenosides enriched fractions were obtained from the total ginsenosides extract of AG roots (TAG) by resin adsorption and heat-transformation. Resin purification decreased the total polyphenol (TP) content in the fractions. However, heat treatment increased the TP content and induced extensive conversion of the polar ginsenosides (Rg1, Re, Rb1, Rc, Rb2, Rd, and Gyp XVII) to less polar compounds (Rg2, Rg3, Rg6/F4, Rs3, and Rg5/Rk1). Only the heat-treated fraction from 65% methanol elution of the HP-20 column (HPHF) showed antibacterial activity against P. acnes (ATCC11827, MIC, 128 μg/mL; ATCC6919 MIC, 64 μg/mL), S. epidermidis (MIC, 4100 μg/mL) and S. aureus (MIC, 15000 μg/mL). In the anti-inflammatory activity assay, 10 μg/mL of HPHF significantly (P < 0.05) inhibited NO production in LPS-stimulated RAW264.7 cells. These results suggest that less polar ginsenosides enriched fraction HPHF obtained from AG might be useful to develop new types of antibacterial substances and new skin care cosmetics for acne prevention and therapy.