Purpose Grapevine (Vitis vinifera L.) is a relevant crop, which is associated to arbuscular mycorrhizal fungi (AMF) that are influenced by agricultural practices. The hypothesis of this study is that organic/biodynamic management stimulates grapevine mycorrhizal colonisation and increases AMF diversity in Chilean vineyards. The aim of this study was to determine the influence of agricultural management on AMF association and AMF diversity in Chilean vineyards. Methods Mycorrhizal colonisation of grapevine roots from organic/biodynamic and conventional vineyards in Northern (Elqui Valley), Central (Casablanca and Cachapoal Valleys), and Southern Chile (Maule and Itata Valleys), was determined under a microscope. AMF diversity was analysed by morphological, and molecular characterisation of spores through SSU-ITS-LSU rRNA region sequence analyses. Results AMF colonisation of grapevine roots was influenced by vineyard management independent of the season. Higher mycorrhizal colonisation was detected in organic/biodynamic grapevine soils (20 - 35%), compared with conventional soils (6 - 31%). Twelve AMF species were identified in vineyards, belonging to five Glomeromycota families. Interestingly, organic/biodynamic vineyards showed higher AMF diversity. The three predominant morphotypes were Funneliformis verruculosum (GL1), Septoglomus sp. (GL4) and Septoglomus constrictum (GL5). Molecular analyses of AMF spores highlighted the occurrence of Septoglomus, Acaulospora, Pacispora and Cetraspora genera in vineyards. Conclusions In this study, AMF diversity in Chilean vineyards is described for the first time. The diversity of AMF in vineyards in Chile was higher than the diversity reported in other wine-producing ecosystems. The understanding of agricultural practices on AMF activity and diversity may be crucial to improve the vineyard management.