Grapevine dieback is an increasing problem in grape vineyards in Algeria. We conducted field surveys each spring between 2006 and 2012 to determine the fungal pathogens associated with grapevine decline in various grape-growing regions. We isolated and identified 3 species of Botryosphaeriaceae (Botryosphaeria dothidea, Diplodia seriata, and Neofusicoccum parvum) from infected wood using a combination of morphological and cultural characters, as well as phylogenetic analyses of ribosomal DNA internal transcribed spacer (ITS) regions and fruiting body. We identified 20 isolates of Diplodia seriata, 13 isolates of Botryosphaeria dothidea, and 7 isolates of Neofusicoccum parvum. Inoculation of grapevine plantlets with the 3 Botryosphaeriaceae species produced necrosis and vascular lesions in the wood after a 6-week incubation; Neofusicoccum parvum isolates were the most virulent, followed by D. seriata and B. dothidea. All 3 species were re-isolated from lesions on infected plantlets. Our results represent the first report of a canker disease of grapevine associated with species of the Botryosphaeriaceae in Algeria.