Colletotrichum acutatum is a major fungal pathogen of fruit crops, which causes severe yield losses in strawberry productions. A potential key factor in plant-pathogen interactions is fungal sesquiterpenoids that have myco- and phytotoxic activities. The first committed step in sesquiterpenoids biosynthesised is performed by sesquiterpene synthases (TPS). Only a few TPS's have been functionally characterized from filamentous fungi and none from the Colletotrichum genus. Despite it as an important fungal pathogen to agriculture, it is poorly understood at the molecular and chemical level. The terpenoid biochemistry in C. acutatum strain SA 0-1 was studied and one C. acutatum TPS (CaTPS) was successfully cloned and characterized in yeast. CaTPS catalyses the biosynthesis of multiple sesquiterpenoids. The two major products are -caryophyllene and an unidentified sesquiterpenoid along with -humulene as one the minor sesquiterpenoid products. These products were also secreted by the fungus in strawberry fruit media along with several other sesquiterpenoids indicating other TPS's are active during in vitro growth. -Caryophyllene and -humulene are known cytotoxic products important for ecological interactions and are produced by SA 0-1. Interestingly, a gene expression analysis using qRT-PCR revealed a significant increase in expression of CaTPS during strawberry fruit infection thus indicating that it could be involved in fruit infection. This is the first characterization of TPS in Colletotrichum spp. and terpenoid profiles of C. acutatum, which could facilitate studies on the role of terpenoids in the ecology of C. acutatum.