Abstract: Soil-borne fungal pathogens cause serious damage to horticultural crops. One of the most serious is Sclerotinia sclerotiorum, whose management relies mainly on chemicals, and more recently on use of biological control agents (BCA). Modern and sustainable disease management strategies for short cycle hort-crops should shift from chemicals to known or new BCA. With the objective to broaden the number of BCA and to get a new insight into the mode of action of streptomycetes able to reduce S. sclerotiorum severity, we have applied an enhanced green fluorescence protein (EGFP) approach to study Streptomyces-mediated biological control. The EGFP gene was integrated through conjugation into five Streptomyces spp. strains that had previously shown strong in vitro antagonistic activity against S. sclerotiorum. The ex-conjugants were selected based on their resistance to apramycin. Conjugation efficiencies varied among strains from 5.81×10-8 to 4.64×10-5 ex-conjugants/ recipient cell. The ex-conjugants fluoresced when observed by fluorescence microscopy, and the presence of the EGFP gene was additionally confirmed by PCR. The influence of EGFP transformation on the growth rate and the in vitro antagonistic activity against S. sclerotiorum was evaluated. None of the characters were significantly altered for the ex-conjugants, except for the EGFP-FT05W strain which showed reduced antagonistic activity. The EGFP-transformed Streptomyces strains will be used to study plant-pathogen-microbe interactions, and will contribute to further understanding of biological control mechanisms of plant soil-borne fungal pathogens.