Antifungal proteins (AFPs) of fungal origin have been described in filamentous fungi. AFPs are small, highly stable, cationic cysteine-rich proteins (CRPs) that are usually secreted in high amounts and show potent antifungal activity against non-self fungi. The role of AFPs in the biology of the producer fungus remains unclear. AFPs have been proposed as promising lead compounds for the development of new antifungals. The analyses of available antifungal CRP sequences from fungal origin and their phylogenetic reconstruction led us to propose a new classification of AFPs in three distinct classes: A, B and C. We initiate for the first time the characterization of an AFP in a fungal pathogen, by analysing the functional role of the unique afpB gene in the citrus fruit pathogen Penicillium digitatum. Null Delta afpB mutants revealed that this gene is dispensable for vegetative growth and fruit infection. However, strains that artificially express afpB in a constitutive way (afpB(C)) showed a phenotype of restricted growth, distortion of hyphal morphology and strong reduction in virulence to citrus fruits. These characteristics support an antifungal role for AfpB. Surprisingly, we did not detect the AfpB protein in any of the P. digitatum strains and growth conditions that were analysed in this study, regardless of high gene expression. The afpB(C) phenotype is not stable and occasionally reverts to a wild type-like phenotype but molecular changes were not detected with this reversion. The reduced virulence of afpB(C) strains correlated with localized fruit necrosis and altered timing of expression of fruit defence genes.