Fungal species in the family Botryosphaeriaceae have been recently recognized as the most common fungi isolated from grapevine (Vitis vinifera) cankers in California. However, the role of these fungi in causing grapevine dieback as well as their status as canker-causing agents was unknown. Therefore, pathogenicity studies were conducted to determine their importance as grapevine pathogens in California. A total of 72 isolates representing all nine Botryosphaeriaceae species isolated from grapevine cankers from California were used in five different pathogenicity studies. Overall, experiments showed all nine Botryosphaeriaceae species able to infect both young and mature tissues as well as green shoots of the new vegetative growth causing cankers, vascular discoloration, and/or otherwise dark streaking of the wood. However, virulence varied among species. Lasiodiplodia theobromae was the most virulent species followed by Neofusicoccum luteum, N. parvum, and N. australe, all categorized as highly virulent. Botryosphaeria dothidea was considered intermediately virulent and Diplodia mutila, D. seriata, Dothiorella iberica, and D. viticola were shown to be weakly virulent. This study shows species of Botryosphaeriaceae to be much more important pathogens on grapevines than originally thought, and some of them, in view of their virulence, should be considered high risk for causing severe and rapid canker and dieback diseases in the grapevine industry in California.