Enniatins (ENs) are secondary metabolites produced by several Fusarium strains, chemically characterized as N-methylated cyclohexadepsipeptides. These compounds are known to act as antifungal and antibacterial agents, but they also possess anti-insect and phytotoxic properties. In this study, the antimicrobial effect of pure fractions of the bioactive compounds ENs A, A₁, A₂, B, B₁, and B₄ was tested towards nine probiotic microrganisms, twenty-two Saccharomyces cerevisiae strains and nine Bacillus subtilis strains. Antimicrobial analyses were carried out the disc-diffusion method using ENs concentrations ranging from 0.2 to 20,000 ng. Plates were incubated for 24 h at 37 °C before reading the diameter of the inhibition spots. ENs A, A₁, A₂, B, B₁ and B₄, were active against several microorganisms with inhibition halos ranging from 3 to 12 mm in diameter. The most active mycotoxin was the EN A₁, which reduced the microbial growth of 8 strains at the dose of 20,000 ng, with inhibition spots sized between 8 and 12 mm. ENs B and B₄ showed no antimicrobial activity towards the microorganisms tested at doses up to 20,000 ng per disc.