Due to their hydrophobic characteristics, hydrophobins function in a broad range of processes in the growth and development of filamentous fungi. In this study, a novel hydrophobin, PN1, was purified from the fruiting bodies of Pleurotus nebrodensis by first removing other proteins from mycelial cell walls with hot SDS solution. The molecular mass of PN1 was approximately 10.7 kDa. The N-terminal AA sequence was QESPVNQCNTGSIEECKTVQ, which did not match that of any other mushroom hydrophobin described thus far. Levels of hydrophobin PN1 protein in the tissue of fruiting bodies was decreased by fruiting body development under low temperatures. Low levels of hydrophobin PN1 was correlated to the formation of water-soaked blotches on fruiting body tissues that appeared dark and translucent, and were usually sunken. In addition, hydrophobin PN1 levels were correlated with protecting fruiting bodies against surface bacterial colonization and multiplication. Low levels of hydrophobin PN1 resulted in water-soaked blotches that had high levels of surface bacteria belonging to multiple species when compared with nonsymptomatic tissue of fruiting bodies with higher hydrophobin PN1. These results show a direct link between mushroom hydrophobins and resistance of mushrooms to bacterial attack.