Aims: To examine the effect of previously described bacterial antagonists on the viability of Verticillium microsclerotia in vitro and to elucidate the possible modes of action of bacterial strains in the suppression of Verticillium microsclerotia viability. Methods and Results: A microplate assay was developed to test the suppressive effect of well-defined Pseudomonas spp. on the viability of Verticillium microsclerotia in vitro. Experiments using phenazine- and biosurfactant-deficient mutants indicated that biosurfactants and phenazine-1-carboxylic acid play a role in the suppression of microsclerotia viability by Pseudomonas spp. In addition, microsclerotia colonization tests revealed that Pseudomonas spp. are able to colonize the surface of the microsclerotia, but not the inner matrix. Growth response curves showed that the population levels of Pseudomonas spp. increased when they were in the vicinity of Verticillium microsclerotia, indicating that Pseudomonas spp. may utilize nutrients from the microsclerotia for their growth. Conclusions: Pseudomonas spp. seem to be good candidates for Verticilllium microsclerotia biocontrol. Biosurfactant production is one of the main mechanisms involved in their mode of action. Significance and Impact of the Study: This line of work may contribute to a better understanding of biological control agents and their working mechanisms.