In the present study, the antibacterial activity of rosemary (Rosmarinus officinalis L. Labiatae) essential oil against Propionibacterium acnes (P. acnes) was observed with atomic force microscopy (AFM). The MIC (minimal inhibitory concentration) value of rosemary essential oil against P.acnes was 0.56 mg/mL. Significant changes in morphology and size of P. acnes were observed by atomic force microscopy (AFM) in response to essential oil treatment. The essential oil first attached to the surface of P. acnes at low concentration, the width and height of the bacterial body became larger, whereas the length did not change considerably. With increasing concentration of the essential oil, the bacterial bodies were severely damaged. The length, width and height were all reduced, when the concentration was increased up to 64xMIC, the length, width and height were reduced by 42.56%, 92.00% and 41.58%, respectively. Furthermore, treated bacteria lost their native shape, the cell wall desquamated, and the cytoplasm leaked out of the bacterial body, finally leading to bacterial death. With the increasing time at MIC, the bacteria length was reduced at 8 h, the width and height gradually became smaller, the shape of the cell became distorted, and finally led to cell wall damage and bacterial death at 8 h. In conclusion, the AFM investigation of morphology and size of P. ACNES treated with rosemary essential oil represents a powerful technique, which can generally be applied to reveal the biological changing mechanisms of bacteria induced by antibacterial agents at the nanometer level.