Antimicrobial peptides are promising therapeutic agents for treating drug-resistant bacterial disease due to their broad-spectrum antimicrobial activity and decreased susceptibility to evolutionary resistance. In this study, three novel cathelicidin antimicrobial peptides were identified from Thamnophis sirtalis, Balaenoptera musculus, and Lipotes vexillifer by protein database mining and sequence alignment and were subsequently named TS-CATH, BM-CATH, and LV-CATH, respectively. All three peptides exhibited satisfactory antibacterial activity and broad antibacterial spectra against clinically isolated E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii in vitro. Among them, TS-CATH displayed the best antimicrobial/bactericidal activity, with a rapid elimination efficiency against the tested drug-resistant gram-negative bacteria within 20 minutes, and exhibited the lowest cytotoxicity toward mammalian cells. Furthermore, TS-CATH effectively enhanced the survival rate of mice with ceftazidime-resistant E. coli bacteremia and promoted wound healing in meropenem-resistant P. aeruginosa infection. These results were achieved through the eradication of bacterial growth in target organs and wounds, further inhibiting the systemic dissemination of bacteria and the inflammatory response. TS-CATH exhibited direct antimicrobial activity by damaging the inner and outer membranes, resulting in leakage of the bacterial contents at super-MICs. Moreover, TS-CATH disrupted the bacterial respiratory chain, which inhibited ATP synthesis and induced ROS formation, significantly contributing to its antibacterial efficacy at sub-MICs. Overall, TS-CATH has potential for use as an antibacterial agent.