Mature canes were collected from vines in the main grapevine‐growing areas in Tunisia (Cape Bon, Bizerte, Ben Arous), from commercial vineyards and mother‐plant plots, to assess the presence of virus and virus‐like diseases. Biological (mechanical transmission onto herbaceous hosts and grafting onto indicator woody plants) and serological detection (ELISA) methods were applied. ELISA showed that 96.4% of 669 vines tested were infected, most of them (88.1%) by at least two viruses. Grapevine leafroll‐associated 3 closterovirus (GLRaV‐3) was the most widespread virus (87.9%), followed by grapevine A vitiviras (GVA, 69.4%), grapevine fleck virus (GFkV, 51.9%), grapevine leafroll‐associated 1 closterovirus (GLRaV‐1, 36.8%), grapevine leafroll‐associated 2 closterovirus (GLRaV‐2, 19.1%), grapevine fan leaf nepovirus (GFLV, 18.2%) and grapevine B vitiviras (GVB, 14.8%). ELISA tests yielded negative results for grapevine leafroll‐associated 7 closterovirus (GLRaV‐7) and potato X potexvirus (PVX). The highest infections were found in Bizerte and Cape Bon regions (100 and 99.2%), and in vineyards aged over 20 years (98.5%) as compared with the younger ones (81.1%). Rootstocks in mother‐plant plots were practically free from all the viruses tested (1 plant infected out of 81), whereas severe infections were found in Vitis vinifera mother plants (67.4% of 341 samples), in particular table grapes (92.6%) compared with wine grapes (47.9%). In these mother‐plant plots, the prevailing viruses were GLRaV‐3 (41.3%), followed by GFkV (36.7%), GVA (27.9%), GLRaV‐1 (17%) and GLRaV‐2 (15.2%). GFLV and GVB were far more limited (1.5 and 0.6%, respectively). The presence of vein necrosis and vein mosaic was ascertained by transmission onto 110R and Vitis riparia indicators, whereas only GFLV was mechanically transmitted onto herbaceous hosts (from about 20% of the samples).