Abstract Severe decline of mature grapevines has recently been observed in several vineyards in grape regions in northern Tunisia. Between August 2011 and June 2013, wood samples from diseased vines showing dead spur and cordons, shoot dieback associated with sunken necrotic bark lesions and brown to black vascular streaking were collected from numerous diseased vineyards. Several fungal species were isolated from the margin between healthy and symptomatic tissue. Three species of Botryosphaeriaceae, namely, Diplodia seriata, Neofusicoccum australe, and N. vitifusiforme and one species of Diaporthaceae, namely, Diaporthe neotheicola, were observed to be associated with the decline of old vines. Other fungal species were recovered from diseased wood, namely, Alternaria alternata, Botryotinia fuckeliana (anamorph of Botrytis cinerea), Acremonium spp., Aspergillus spp., and Fusarium spp. In addition, Penicillium spp. inter- and intra-species diversity were assessed based on virtual RFLP gel analyses and identification of restriction enzymes able to distinguish fungi strains within species based on determination of single nucleotide polymorphism (SNP) lineages within cluster members based on the sequencing of the internal transcribed spacer (ITS1-5.8S-ITS2) region of the rDNA.