Symbiotic bacteria, which are carried in the intestinal vesicle of the infective stage of juvenile ento- mopathogenic nematodes, produce broad-spectrum antibiotics. In this study, we aimed to isolate the antimicrobial peptides from the culture of the entomopathogenic bacterium Xenorhabdus budapestensis NMC-10. By screening chromatography columns and optimizing flow rate, pH, salinity and other purifi- cation conditions, we identified the final purification procedures which consisted of Q ion-exchange chromatography, gel filtration chromatography and two-step reverse-phase chromatography. Two novel antimicrobial peptides were identified via Q-TOF-TOF and de novo sequencing, and designated as GP-19 and EP-20. Both natural and synthetic peptides demonstrated broad-spectrum antimicrobial activities. The synthetic GP-19 peptide was active against Verticillium dahlia with EC50 values of 17.54 g/ml and highly inhibited the growth of a variety of bacteria, while the synthetic EP-20 peptide was highly active against Phytophthora capsici with EC50 values of 3.14 g/ml