The grey mould fungus Botrytis cinerea is a typical necrotrophic fungus that can infect hundreds of host plants, including high-value crops such as grapevine, strawberry and tomato. In order to find new important genes involved in the fungal virulence, a mutant library was generated by random insertional mutagenesis, using Agrobacterium tumefaciens mediated transformation. Ten mutants exhibiting total loss of virulence towards different host plants were considered. Their molecular characterization revealed a single T-DNA insertion in unique and different loci. Using a proteomics approach, the secretome of four of these strains was compared to that of the parental strain and a common profile of reduced lytic enzymes was recorded. Significant variations in this profile, notably proteases and hemicellulases deficiencies, were observed and validated by biochemical tests. They were also a hallmark of the remaining six non pathogenic strains, suggesting the importance of these secreted proteins in the infectious process.