Antimicrobial peptides (AMPs) have gained interesting as a new type of antimicrobial agent. The cathelicidin-derived antimicrobial peptide PMAP-23 has broad-spectrum antibacterial activity, and to improve its antimicrobial activity, we used amino acid substitution at position 5 or 19 of PMAP-23 to design three analogs, named PMAP-23R (Leu--Arg), PMAP-23I (Thr--Ile), and PMAP-23RI (Leu--Arg and Thr--Ile). We found that the analog peptides exhibited higher stability and improved antibacterial activity compared with PMAP-23. Additionally, the analog peptides PMAP-23I and PMAP-23RI inhibited the growth of Shigella flexneri CICC 21534, whereas PMAP-23 and PMAP-23R exhibited no antibacterial activity against S. flexneri CICC 21534. Moreover, the peptide analogs showed negligible hemolysis and cytotoxicity. We also found that PMAP-23RI exerted impressive therapeutic effects on mice infected with Staphylococcus aureus ATCC 25923 and Salmonella enterica serovar Typhimurium SL1344. PMAP-23RI induced a greater reduction in pathological damage and a higher decrease in the bacterial gene copies in the lung and liver tissues and greatly reduced mouse mortality. In conclusion, the peptide analogs PMAP-23R, PMAP-23I, and PMAP-23RI enhanced the stability and antimicrobial activity of PMAP-23, but PMAP-23RI exhibits more promise as a new antimicrobial agent candidate for the treatment of bacterial infections.