Black cumin (Nigella sativaL.) is known to possess a wide variety of antimicrobial peptides belonging to different structural families. Three novel antimicrobial peptides have been isolated from black cumin seeds. Two of them were attributed as members of the non-specific lipid transfer proteins family, and one as a defensin. We have made an attempt of using the proteomic approach for novel antimicrobial peptides search inN. sativaseeds as well. The use of a well-established approach that includes extraction and fractionation stages remains relevant even in the case of novel peptides search because of the lackingN. sativagenome data. Novel peptides demonstrate a spectrum of antimicrobial activity against plant pathogenic organisms that may cause economically important crop diseases. These results obtained allow considering these molecules as candidates to be applied in “next-generation” biopesticides development for agricultural use.