Historically, plant-parasitic nematodes (PPNs) have been an important pathogen of grapevine (Vitis vinifera L.) worldwide. In this study, we tested the hypothesis that PPNs are associated with grapevine decline disease and/or that they are potential pathogens. The community structure of PPNs was assessed based on morphological and morphometric measures. During the 2010/2011 and 2016/2017 seasons, soil and root samples were collected from 64 vineyards and two nurseries in the South of Brazil. We found large taxonomic diversity in the vineyards, with the species recorded more frequently belonging to the genera Helicotylenchus, Mesocriconema, Xiphinema, and Hemicycliophora. In the nurseries, the most frequent and abundant PPNs were Meloidogyne (M. incognita, M. javanica, M. arenaria, M. morocciensis and M. luci), Pratylenchus (P. brachyurus and P. zeae), Mesocriconema and Helicotylenchus. To our knowledge, this is the first report of PPNs community in Brazilian grapevine areas with the Decline disease and the first record of M. luci associated with grapevine in Brazil. Additional studies are needed evaluate resistance of rootstocks to the main PPNs associated with Decline disease in Brazil.