Abstract: The reduction of the rate of ascospore inoculum of Venturia inaequalis in apple cultivations can facilitate scab control and reduce the risk of selection of pathogen-resistant populations to fungicides. Potential candidate antagonistic fungi were isolated from apple leaves on the soil of several natural areas in Italy and tested against conidial germination and mycelial growth of V. inaequalis, in vitro. The most effective isolates were further assayed for their eradicant activity on ascospores of an apple orchard, both alone and in mixture with additive substances (glycerol and glucose) and applied at two different timings: at the end of leaf-fall or at the beginning of pseudothecia maturation. The antagonistic activity of these isolates was evaluated by assessing the number of ascospores extracted from leaves by a “wind tower”. Several fungal isolates (Penicillium spp., Acremonium spp. and Verticillium spp.) caused a reduction of the number of ascospores greater than 88%, in the first or in the second application time, and there effect was positively influenced by the additive substances. The eradicant activity of several antagonistic fungi is promising and further trials are in progress to confirm these results and to study their modes of action.