Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics, resulting in high mortality rates of 19% in Australia and even 50% in Thailand. Antimicrobial peptides (AMPs) possess potent broad-spectrum bactericidal activities and are regarded as promising therapeutic alternatives in the fight against resistant microorganisms. Moreover, these peptides may also affect inflammation, immune activation and wound healing. In this study, the in vitro activities of 10 AMPs, including histatin 5 and histatin variants, human cathelicidin peptide LL-37 and lactoferrin peptides, against 24 isolates of B. pseudomallei were investigated. The results showed that the antibacterial activities of the individual peptides depended on peptide dose and bacterial isolate. Among the 10 peptides tested, LL-37 exhibited the most effective killing activity. The smooth type A lipopolysaccharide (LPS) phenotype B. pseudomallei appeared to be more susceptible than those expressing the smooth type B LPS and the rough type LPS. Four isolates of B. pseudomallei shown to be resistant to ceftazidime and trimethoprim/sulfamethoxazole were also highly susceptible to LL-37. These data indicate that LL-37 possesses antimicrobial activity against all isolates independent of the LPS phenotype and is therefore a promising peptide to combat B. pseudomallei infections.