Muscadine ( Muscadinia rotundifolia ) is known as a resistance source to many pests and diseases in grapevine. The genetics of its resistance to two major grapevine pests, the phylloxera D. vitifoliae and the dagger nematode X. index , vector of the Grapevine fanleaf virus (GFLV) was investigated in a backcross progeny between the F1 resistant hybrid material VRH8771 ( Muscadinia-Vitis ) and V. vinifera cv. ‘Cabernet-Sauvignon’. Phylloxera resistance was assessed by monitoring root nodosity number in an “ in planta ” experiment and larval development in a root “ in vitro ” assay. Nematode resistance was studied using 10-12 month long tests for the selection of durable resistance and rating criteria based on nematode reproduction factor and gall index. A major QTL for phylloxera larval development, explaining more than 70% of the total variance and co-localizing with a QTL for nodosity number, was identified on LG 7. Additional QTLs were detected on LG 3 and LG 10, depending on the “ in planta ” or “in vitro” experiments, suggesting that various loci may influence or modulate nodosity formation and larval development. Using a Bulk Segregant Analysis approach and a proportion test, markers clustered in three regions on LG 9, LG 10 and LG 18 were shown to be associated to the nematode resistant. All these results open the way to the development of marker-assisted selection in grapevine rootstock breeding programs based on muscadine derived resistance to phylloxera and to X. index in order to delay GFLV transmission.