A novel method named cell membrane affinity chromatography was used to screen antimicrobial peptides from Jatropha curcas . A cationic antimicrobial peptide (KVFLGLK, JCpep7) was successfully isolated and identified. Antimicrobial assays indicated that JCpep7 was active against the tested microorganisms ( Salmonella typhimurium ATCC 50013, Shigella dysenteriae ATCC 51302, Pseudomonas aeruginosa ATCC 27553, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 23631, and Streptococcus pneumoniae ATCC 49619) with minimal inhibitory concentration (MIC) values ranging from 24 to 64 μg/mL. The antimicrobial mechanisms based on Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM) techniques showed that JCpep7 killed microbes principally via breaking of their cell walls and membranes, followed by cell lysis. The results indicated that cell membrane affinity chromatography could be a promising approach for high-throughput screening of antimicrobial peptides from J. curcas .