Antibiotic resistance has become a significant global public health challenge, particularly in general surgery, where infections caused by resistant bacteria complicate treatment. This study aims to evaluate the potential of the FDA-disapproved antihistamine astemizole as an antibacterial agent, with a focus on its efficacy against methicillin-resistant S. aureus (MRSA). Astemizole demonstrated significant activity against Gram-positive bacteria, especially MRSA, with MIC and MBC values ranging from 4 to 16 μg/mL and 4-32 μg/mL, respectively. However, astemizole showed minimal activity against Gram-negative bacteria. Further investigations revealed that astemizole killed bacteria by disrupting the bacterial membrane, altering membrane potential, inhibiting ATP production, and inducing reactive oxygen species accumulation. Additionally, The resistance mutation frequency of astemizole was low, with only a minor increase in resistance observed in MRSA after 30 days of selective pressure, significantly less than that of ampicillin. Cytotoxicity and hemolysis assays indicated that astemizole was relatively safe at concentrations effective for bacterial inhibition. The Galleria mellonella infection model further confirmed the efficacy of astemizole against MRSA in vivo. Overall, this study provides new insights into the repurposing of astemizole and suggests its potential as a therapeutic agent to address antibiotic resistance.