The number of cationic residues and net charge are critical for the activity of antimicrobial peptides (AMPs) due to their role in facilitating initial electrostatic interactions with negatively charged bacterial membranes. A cathelicidin AMP (TC-33) has been identified from the Chinese tree shrew in our previous work, which exhibited weak antimicrobial activity, likely due to its moderately cationic nature. In the current study, based on TC-33, we designed a novel AMP by peptide truncation and Glu substitutions to increase its net cationic charge from +4 to +8. The resulting peptide, TC-LAR-18, showed 4-128-fold enhanced antimicrobial activity relative to TC-33 without causing hemolysis and cytotoxicity within 100 mu g/mL. TC-LAR-18 effectively eliminated both planktonic and biofilm-associated bacteria, demonstrating rapid bactericidal effects due to its ability to quickly penetrate and disrupt bacterial cell membranes with a low propensity to induce resistance. Notably, TC-LAR-18 provided substantial protection against skin bacterial infection in mice, underscoring its therapeutic potential. These findings not only highlight the importance of positively charged residues for the antibacterial activity of AMPs but also present a useful drug candidate for combating multidrug-resistant bacteria.