Abstract: The recently identified phylogenetic subgroup B5 of fungal glycoside hydrolasefamily 18 genes encodes enzymes with mannosyl glycoprotein endo-N-acetyl-β-Dglucosaminidase(ENGase)-type activity. In the current study, the Trichoderma atrovirideEng18B ENGase gene was deleted and the resulting phenotypes studied, with emphasis on itsrole in fungal growth and antagonism. Eng18B deletion strains had significantly reduced growthrates but higher conidiation rates compared to the wild-type strain. However, growth rates onabiotic stress media were significantly higher in Eng18B deletion strains compared to the wildtypestrain. In addition, we determined that Eng18B is required for the antagonistic ability ofT. atroviride against the grey mould fungus Botrytis cinerea in dual cultures and that thisreduction in antagonistic ability is partly connected to a secreted factor. The phenotypes wererecovered by re-introduction of an intact Eng18B gene fragment in mutant strains. A putative roleof Eng18B ENGase activity in the endoplasmic reticulum associated protein degradation pathwayof endogenous glycoproteins in T. atroviride is discussed in relation to the observed phenotypes.