To produce enniatin (ENs), Fusarium tricinctum CECT 20150 was grown in a liquid medium of potato (PDB), being mycotoxin purified by high performance liquid chromatography (HPLC) with a reverse phase semipreparative column using a mobile phase of acetonitrile/water using gradient condition. The purity of the ENs fractions was verified by analytical HPLC and LC/MS-MS. The pure fractions of ENs were utilized to study the biological activity on several mycotoxigenic moulds as Fusarium verticilloides, Fusarium sporotrichioides, Fusarium tricinctum, Fusarium poae, Fusarium oxysporum, Fusarium proliferatum, Beauveria bassiana, Trichoderma harzianum, Aspergillus flavus, Aspergillus parasiticus, Aspergillus fumigatus, Aspergillus ochraceus and Penicillium expansum.The results obtained demonstrated that in several antibiograms, ENs induced the inhibition of the grown microorganisms tested.