The antimicrobial activity and the underlying action mechanisms of dihydrotanshinone I against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamases Staphylococcus aureus were investigated with Kleihauer-Betke (K-B) test. The antibacterial mechanisms of dihydrotanshinone I were investigated by monitoring the changes in electric conductivity, concentration of AKP, protein content, and patterns of protein electrophoretic bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The antibacterial rings showed that antimicrobial activity of dihydrotanshinone I at 18 mM was stronger to Staphylococcus aureus than to methicillin-resistant Staphylococcus aureus and extended-spectrum beta-lactamases Staphylococcus aureus. The minimum inhibitory concentration (MIC) and IC50 values showed that dihydrotanshinone I had the strongest inhibitory activity against S. aureus (MIC = 280 µM, IC50 = 874 ± 0.01 µM, respectively). Dihydrotanshinone I could increase the electric conductivity, concentration of alkaline phosphatase (AKP) and protein content. The patterns of protein bands in SDS-PAGE were changed obviously. Dihydrotanshinone I also significantly inhibited S. aureus, methicillin-resistant S. aureus, and extended-spectrum beta-lactamases S. aureus, indicating that dihydrotanshinone I can damage the structures of cell wall and cell membrane to increase permeability of cell membrane and release of cell components. Dihydrotanshinone I could influence the synthesis of bacterial protein, destroy the protein, or reject the anabolism or expression of the protein, and finally lead to the loss of normal physiological function of bacteria.