The transcription factor THCTF1 fromTrichoderma harzianum, previously linked to the production of 6-pentyl-2H-pyran-2-one (6-PP) derivatives and antifungal activity againstFusarium oxysporum, has been related in this study to conidiation, production of an array of volatile organic compounds (VOCs) and expression of methyltransferase genes. VOCs emitted by threeT. harzianumstrains (wild type T34, transformant ΔD1-38 that is disrupted in theThctf1gene encoding the transcription factor THCTF1, and ectopic integration transformant ΔJ3-16) were characterized by Proton Transfer Reaction-Quadrupole interface-Time-Of-Flight-Mass Spectrometry (PTR-Qi-TOF-MS).Thctf1disruption affected the production of numerous VOCs such as the antifungal volatiles 2-pentyl furan and benzaldehyde which were under-emitted, and acetoine, a plant systemic defense inductor, which was over-emitted. Biological assays show that VOCs regulated by THCTF1 are involved in theT. harzianumantifungal activity againstBotrytis cinereaand in the beneficial effects leading toArabidopsisplant development. The VOC blend from the disruptant ΔD1-38: (i) inhibitedArabidopsisseed germination for at least 26 days and (ii) when applied toArabidopsisseedlings resulted in increased jasmonic acid- and salicylic acid-dependent defenses.