Pathogenic fungi and bacteria infect plants and inflict important diseases leading to major economic losses. The use of beneficial Lactic Acid Bacteria (LAB) in controlling different type of human and plant pathogens, has drawn worldwide attention. The aim of this study was to assess the antifungal activity of secreted substances from two newly isolated strains of Enterococcus faecium (Kx185054 and Kx185055) from Persian oak tree sap in controlling Fusarium oxysporum and Alternaria solani fungi and Curtobacterium flaccumfaciens pv. flaccumfaciens and Pseudomonas syringae pv. tomato bacteria. Secreted substances from Enterococcus faecium strains were added to the fungi and bacteria culture media at different concentrations and the inhibition zone was recorded in three replicates. Results of this experiments together with that of scanning electron microscopy imaging revealed that secreted substances had a significant antimicrobial activity. Fungal mycelia/spores and bacterial cells became deformed, shrunken, and distorted following treatment with secreted substances from Enterococcus faecium strains, suggesting that Enterococcus faecium secretes antimicrobial chemicals/peptides preventing fungal strictures from germination, growth and penetration. The results of this study may help in elucidating the potentials of newly isolated Enterococcus faecium strains from oak tree and identifying the genes coding for antifungal peptides.