Plant disease control is dominated by an over-reliance on chemical pesticides, in order to increase yield and quality. Since at least the 1930s, fungi of several genus have been known to be able to control soil-borne pathogens. A number of isolates of different species recovered from soil, rhizosphere and directly from colonies developing onto Rhizoctonia cultures, were screened in laboratory, greenhouse and plot tests. In vitro inhibition tests were carried out in Petri dishes and consisted in dual cultures and addition of culture filtrates to evaluate the presence of toxic metabolites. In vivo assays were carried out in the greenhouse assessing disease incidence and severity on tobacco plants grown in soil inoculated with Rhizoctonia solani in the presence of tested isolates; in the greenhouse and in the field, a number of biometrical parameters were measured to evaluate plant growth promotion. Seven isolates, belonging to Verticillium biguttatum (2 isolates), V. psalliotae , Gliocladium roseum , Trichoderma aureoviride , Penicillium oxalicum , P. pinophilum showed to have some efficacy against Rhizoctonia solani . Application of antagonist alone did not affect plant development, some of them exhibited even plant growth promotion. Some of proposed isolates showed some interesting features to be developed as biocontrol agents in tobacco production.