Peptaibols (Paib), are a class of biologically active peptides isolated from soil, fungi and molds, which have interesting properties as antimicrobial agents. Paib production was optimized in flasks by adding sucrose as a carbon source, Aib as an additive amino acid, and F. oxysporum cell debris as an elicitor. Paib were purified, sequenced and identified by HPLC coupled to mass spectrometry. Af-terward, a Paib prototype was prepared with the extracts obtained from the optimized fermenta-tions. The biological activity of these prototypes was evaluated using in vitro and in vivo methods. The prototype inhibited the growth of specific plant pathogens, and it showed inhibition rates similar to those from commercially available fungicides. Growth inhibition rates were 92.2, 74.2, 58.4 and 36.2 % against Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria alternata and Fusarium oxysporum, respectively. Furthermore, the antifungal activity was tested in tomatoes inoculated with A. alternata, the incidence of the disease in tomatoes treated with the prototype was 0%, while the untreated fruit showed a 92.5% of infection. Scanning electron microscopy images showed structural differences between fungi treated with or without Paib. The most visual altera-tions were sunk and shriveled morphology in spores, while the hyphae appeared to be fractured, rough, and dehydrated.