Grapevine trunk diseases (GTDs), caused by fungal pathogens, are a serious threat to vineyards worldwide, causing significant yield and economic loss. To date, curative methods are not available for GTDs, and the relationship between the pathogen and symptom expression is poorly understood. Several plant pathologists, molecular biologists, and chemists have been investigating different aspects of the pathogenicity, biochemistry, and chemical ecology of the fungal species involved in GTDs. Many studies have been conducted to investigate virulence factors, including the chemical characterization of phytotoxic metabolites (PMs) that assist fungi in invading and colonizing crops such as grapevines. Moreover, multidisciplinary studies on their role in pathogenicity, symptom development, and plant-pathogen interactions have also been carried out. The aim of the present review is to provide an illustrative overview of the biological and chemical characterization of PMs produced by fungi involved in Eutypa dieback, Esca complex, and Botryosphaeria dieback. Moreover, multidisciplinary investigations on host-pathogen interactions, including those using cutting-edge Omics techniques, will also be reviewed and discussed. Finally, challenges and opportunities in the role of PMs for reliable field diagnosis and control of GTDs in vineyards will also be explored.