Cationic antimicrobial peptides (AMPs) are consideredas important candidate therapeutic agents, which exertpotent microbicidal properties against bacteria, fungiand some viruses. Based on our previous findingsking cobra cathelicidin (OH-CATH) is a 34-aminoacid peptide that exerts strong antibacterial andweak hemolytic activity. The aim of this researchis to evaluate the efficacy of both OH-CATH30 andits analog D-OH-CATH30 against clinical isolatescomparing with routinely utilized antibiotics in vitro.In this study, 584 clinical isolates were tested(spanning 2013–2016) and the efficacy of thecandidate peptides and antibiotics were determinedby a broth microdilution method according tothe CLSI guidelines. Among the 584 clinicalisolates, 85% were susceptible to OH-CATH30and its analogs. Both L- and D-OH-CATH30showed higher efficacy against (toward) Gram-positivebacteria and stronger antibacterial activity againstnearly all Gram-negative bacteria tested comparewith antibiotics. The highest bactericidal activitywas detected against Acinetobacter spp., includingmulti-drug-resistant Acinetobacter baumannii (MRAB)and methicillin-resistant Staphylococcus aureus(MRSA). The overall efficacy of OH-CATH30 and itsanalogs was higher than that of the 9 routinely usedantibiotics. OH-CATH30 is a promising candidatedrug for the treatment of a wide variety of bacterialinfections which are resistant to many routinely usedantimicrobial agents.