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.