Objective To explore the in vitro and in vivo antibacterial activity of acetazolamide against vancomycin resistant enterococci, and investigate its possible mechanism of action.Methods The study object was Enterococcus (N1520), which was clinically isolated from the hospital and identified as vancomycin resistant Enterococcus (VRE). The antibacterial activity in vitro and in vivo were studied respectively. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of acetazolamide on VRE using agar plate dilution method. Determination of the Time Sterilization Change Curve of the Strain to be Tested through the Sterilization curve experiment. Detection of extracellular alkaline phosphatase (AKP) content to evaluate the destructive effect on cell walls, calculation of relative conductivity measurement to evaluate cell membrane permeability. Establishing a mouse model of peritonitis using clinically isolated VRE strains, divided into a control group (normal mice), a model group (peritonitis mice), and an acetazolamide group (30mg/kg/5h intervention). After 30 hours of intervention, the death status of each group of mice was counted, and bacterial colonies in peritoneal fluid, spleen, and liver were counted. HE staining was used to observe pathological changes in the spleen and liver. Results The antibacterial activity test of acetazolamide on VRE showed that MIC and MBC were 1.45 mg/L and 4.62 mg/L, respectively. Time Kill curve analysis showed that the bactericidal activity of acetazolamide was positively correlated with time and concentration. The higher the concentration of acetazolamide, the higher the bacterial mortality rate. The longer the time, the more bacteria were killed. When the concentration of acetazolamide reaches 16 × MIC, when the action time reaches 12 hours, the growth of VRE bacteria was completely inhibited. After 30 hours of intervention, the survival rate of the blank control group was 100%, the model group was 30%, and the acetazolamide group was 80%. The survival rate of mice in the acetazolamide group was significantly higher than that in the model group, with a statistically significant difference (P＜0.05). Compared with the blank control group, the bacterial load in the peritoneal fluid, liver, and spleen of the model group mice was significantly increased, with a statistically significant difference (P＜0.05). Compared with the model group, the bacterial load in the peritoneal fluid, liver, and spleen of the acetazolamide group mice were significantly reduced, with a statistically significant difference (P＜0.05). Compared with the control group, the AKP content in the 1×MIC group and 4×MIC group significantly increased (P＜0.05). Consistent with 1×MIC group comparison, the AKP content in the 4×MIC group significantly increased (P＜0.05), and the destructive effect of acetazolamide on VRE cell wall showed a dose-dependent manner. Compared with the control group, the conductivity of 1×MIC group and 4×MIC group significantly increased (P＜0.05). And with 1×MIC group comparison, the relative conductivity of 4×MIC group significantly increased (P＜0.05), and the increase in VRE cell permeability by acetazolamide showed a dose-dependent effect.Conclusion Acetazolamide has good antibacterial activity against Enterococcus in vivo and in vitro, and its mechanism may be related to the destruction of cell integrity