Antimicrobial peptides (AMPs) possess broad-spectrum antibacterial properties and low resistance development, making them promising candidates for new antibacterial drugs. Incorporating fatty acid chains into AMPs can increase their hydrophobicity, strengthen membrane affinity, and improve their antibacterial effectiveness and stability. This study introduces fatty acid chains of varying lengths into the naturally derived antimicrobial peptide B1. These modified peptides were evaluated for their antibacterial activity, stability, and biocompatibility to identify the optimal chain length for analogues. The analogues B1-C6 and B1-C8 exhibited significantly enhanced antimicrobial activities against Staphylococcus aureus (S. aureus) and Klebsiella pneumoniae (K. pneumonia), demonstrating better stability and biocompatibility. Following acute toxicity and skin irritation tests on mice, further in vivo tests using a mouse skin inflammation model showed that these peptides significantly restrain bacterial growth and promote wound healing. The skin healing rate in the high-concentration group reached 95.92%, 97.35% 98.42% and 98.17%, respectively. These findings indicated that optimizing the hydrophobic-hydrophilic balance in AMPs is crucial for maximizing their therapeutic potential. This research offers a promising approach for designing effective AMPs to treat infections caused by S. aureus and K. pneumoniae.