Aegerolysins are small secreted pore-forming proteins that are found in both prokaryotes and eukaryotes. The role of aegerolysins in sporulation, fruit body formation, and in lysis of cellular membrane is suggested in fungi. The aim of the present study was to characterize the biological function of the aegerolysin gene agl1 in the mycoparasitic fungus Trichoderma atroviride , used for biological control of plant diseases. Gene expression analysis showed higher expression of agl1 during conidiation and during growth in medium supplemented with cell wall material from the plant pathogenic fungus Rhizoctonia solani as the sole carbon source. Expression of agl1 was supressed under iron-limiting condition, while agl1 transcript was not detected during T . atroviride interactions with the prey fungi Botrytis cinerea or R . solani . Phenotypic analysis of agl1 deletion strains (Δ agl1 ) showed reduced conidiation compared to T . atroviride wild type, thus suggesting the involvement of AGL1 in conidiation. Furthermore, the Δ agl1 strains display reduced antagonism towards B. cinerea and R . solani based on a secretion assay, although no difference was detected during direct interactions. These data demonstrate the role of AGL1 in conidiation and antagonism in the mycoparasitic fungus T . atroviride .