Objective To explore the in vitro and in vivo antimicrobial activity of pinaverium bromide (PVB)against Staphylococcus epidermidis (S. epidermidis). Methods S. epidermidis isolated from blood specimens of hospitalized patients in a hospital in Changsha from January to December 2022 were collected. Susceptibility test of S. epidermidis to PVB was performed using broth microdilution method and disc diffusion method. The time- and concentration-dependent antimicrobial activity of PVB were determined by time-killing assay. Ultrastructural changes in bacteria after PVB treatment was observed by transmission electron microscope. The inhibitory and clearance effects of PVB on S. epidermidis biofilm were detected by crystal violet staining test. The combined antimicrobial effect of PVB and antimicrobial agents was studied through microdilution checkerboard technique. A skin abscess infection model was constructed to detect the in vivo antimicrobial activity of PVB. Results Antimicrobial susceptibility testing results showed that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PVB against the standard strains RP62A and ATCC 12228 were 8 and 16 μg/mL, respectively; The MIC and MBC of clinical strains of S. epidermidis were 4-8 μg/mL and 8-16 μg/mL, respectively. Disc diffusion method results showed that compared with the untreated control group (0.60±0) cm, 0.2 mg PVB treatment showed a significant inhibitory zone (［2.26±0.09］ cm; t=45.34, P<0.001), and the diameter of inhibitory zone increased with the increase of PVB dosage. The time-killing curves indicated PVB had bactericidal activity, which enhanced with increased concentration and action duration. Transmission electron microscope observed that PVB could significantly damage the normal structure of S. epidermidis, leading to bacterial edema and lysis. In addition, at the concentration of 1×MIC, PVB could significantly inhibit the formation of S. epidermidis biofilm, reducing the amount of biofilm formation (A_{570 nm}) from (2.30±0.18) to (0.47±0.11; t=14.85, P<0.001). Meanwhile, PVB at the concentration of 1×MIC could effectively destroy the formed biofilm, reducing the amount of biofilm from (2.64±0.10) to (1.77±0.30; t=4.76, P=0.009). The combination of PVB with amikacin and gentamicin exhibited synergistic antimicrobial activity, with synergistic inhibitory indexes of 0.50 and 0.31, respectively. Animal models showed that 10 mg/kg body weight of PVB could reduce the area of abscesses from (68.83±10.68) mm² to (35.50±10.58) mm² (t=6.52, P<0.001), and reduce the amount of viable bacteria in abscesses from (6.11±0.55) lg (CFU/abscess) to (3.60±0.34) lg (CFU/abscess) (t=3.08, P=0.014). Hematoxylin-eosin staining revealed that the infiltration of inflammatory cells in skin abscesses in the PVB treatment group reduced significantly compared with the control group, tending to be normal. Conclusion PVB exhibits effective in vitro and in vivo antimicrobial effect against S. epidermidis, which can be used as an alternative for the treatment of S. epidermidis-related infections.