The cultivation of grapevine, one of the most important agricultural species in Croatia, is often endangered by various diseases caused by viruses, consequently affecting yield and fruit quality. Ecologically, one of the acceptable strategies for increasing a plant's resistance to different environmental stresses is using arbuscular mycorrhizal fungi (AMF) in cultivation. This study aims to determine the consequences of grapevine, AMF, and viruses interactions on grapevine primary metabolism and establish the potential role of AMF in grapevine defence response against viruses. The study was conducted on Merlot grapevine infected with three widespread and/or economically important viruses (GRSPaV, GLRaV-3, GPGV) and inoculated with AMF. It included 15 treatments with different combinations of grapevine, virus and AMF. Enzyme activities involved in glycolysis, pentose-phosphate pathway, starch biosynthesis and sucrose metabolism were analysed using spectrophotometric semi-high throughput methods. The interaction of grapevine, viruses and AMF caused changes in the activity of some enzymes involved in the carbohydrate metabolism of grapevine leaves. Virus infection had a minor impact on carbohydrate metabolism, and certain combinations of viruses affected metabolic enzymes differently. Inoculation with AMF increased sucrose synthesis and decreased starch synthesis, as well as the activities of enzymes involved in glycolysis in uninfected and infected grapevine leaves. Study results contribute to a better understanding of the role of primary metabolism in the interaction of AMF, virus and grapevine.