The aim of this study was to evaluate the antibacterial activity and underlying mechanism of ethanol extracts of Meconopsis quintuplinervia Regel (EMQ) against the acne-causing bacteria Propionibacterium acnes and Staphylococcus aureus. The study results indicated that EMQ was an effective antibacterial agent against P. acnes and S. aureus, with a DIZ of 14.5 and 13.2mm, MIC of 12.5 and 12.5mg/mL and MBC of 100 and 50mg/mL, respectively. EMQ induced morphological changes to bacterial cells, as determined by electron microscopy. Leakage of alkaline phosphatase and nucleic acids confirmed that EMQ compromised the membrane integrity of bacterial cells. Furthermore, protein analysis revealed that EMQ hindered total protein expression and lowered adenosine triphosphatase activity, while crystal violet staining revealed suppressed biofilm production. Bacterial adhesion analysis demonstrated that EMQ lowered the adhesive capacity of bacterial cells. The main chemical components of EMQ, identified by LC-MS, seem to have important roles in the antimicrobial effects against P. acnes and S. aureus, suggesting EMQ is a promising therapeutic for acne treatment.