To adapt to external stimuli, bacteria fine-tune important protein activities using post-translational modifications. The present study provides novel insights into the molecular mechanism of the antimicrobial peptide BCp12. We demonstrate that BCp12 significantly suppressed bacterial growth, induced cell apoptosis, and modulated overall malonylation levels in Staphylococcus aureus cells. Malonylateomic analysis was performed to identify the proteins malonylated by the BCp12 treatment of S. aureus. In total, 53 malonylated proteins (17 up-regulated, 36 down-regulated) were identified as differentially expressed malonylated proteins (DMPs; > 1.5-fold or <0.67-fold, P < 0.05). This result was confirmed via the identification of 21 differential metabolites (DMs; VIP > 1, P < 0.05) in the arginine and proline metabolome. Bioinformatic analysis revealed that the DMPs and DMs were especially enriched in the arginine synthesis pathway. By integrating our lysine malonylational and metabolomic data, we provide new insights into the mechanism by which BCp12 inhibits S. aureus.