Abstract: 9-Acetoxycycloberberine (1) with a unique skeleton was first identified to display a potent antimicrobial profile against methicillin-resistant Staphylococcus aureus (MRSA) with MIC values of 1-16 μg·mL^-1. Taking the compound as a lead, 14 target cycloberberine analogues with diverse structures, such as berberine and chelerythrine derivatives, were synthesized and evaluated for their anti-bacterial activities. Analysis of the structure-activity relationship revealed that:① ring E was essential for the activity; ② the removing of ring B decreased the activity against MRSA. However, the antimicrobial activity against vancomycin-resistant Enterococcus faecium (VRE) was improved; ③ the introduction of a suitable rigid substituent at the 9-position was beneficial for the activity. Among them, compound 9a showed the most potential activity against methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA isolates with MIC values of 0.5-1 μg·mL^-1, suggesting a different mechanism from clinical drugs. It displayed higher stability in blood. Therefore, we consider 9a worthy of further investigation. The results provide key scientific evidence for development of such compounds into a new type of anti-MRSA candidates.