Various fungicides are used to protect different crops from pathogenic fungi attacks, but very little is known about the damaging effects of fungicides’ application on non-target, beneficial soil microflora. Laboratory experiments were conducted to study the effects of Benlate fungicides on the antimicrobial activities of eight antimicrobial strains of Bacillus and E. coli isolated from the Kingdom of Saudi Arabia. The Bacillus and E. coli strains were exposed to 0.5% Benlate, then tested for their inhibition effects of the pathogenic fungi that cause wound inflammations and ulcers, i.e., Acremonium sp., Microsporium gypseum, Microsporium gallinae, Exserohilum sp. and A. niger. Their DNAs were also analyzed for any changes using RAPD-PCR. Results showed different effects of Benlate on bacterial antagonistic activities including more and less antagonistic efficiencies against target pathogenic fungi. Random amplified polymorphic DNA (RAPD) technique was used to check for any changes in DNA after their treatment with Benlate. Different changes in the genome was indicated including variation in band intensity; loss and appearance of new DNA bands. In conclusion, results revealed that treatment with Benlate induced great changes of genomic DNA of non-target bacteria and in their physiological activations. In addition, the results demonstrated that RAPD technique is useful for detection of alterations in the DNA. We observed the inhibitory effect of Bacillus strains and E. coli M2 on pathogenic fungi; however, what molecule was endowed with antifungal activity is not yet known. Further studies are needed to identify and characterize the antifungal protein molecule (s) of Bacillus and E. coli having potential against fungi. The work on identification of the molecule endowed with antimycotic properties is in progress.