Flavescence dorée (FD) has been recorded in French vineyards since the mid-1950s; it has rapidly become a major threat to vineyard survival in different European grape-growing areas. Infection by the phytoplasma ‘Candidatus Phytoplasma vitis’ causes leaf chlorosis and disturbs primary and secondary core metabolic pathways; it can also occlude sieve plates, therefore affecting normal solute transport through the phloem. Thus, in the present study we hypothesised that the metabolomic fingerprint of the phloem sap changes in FDp-infected vines. The metabolome of the phloem sap collected by centrifugation from the bark of the stem of healthy and infected vines (cv. ‘Loureiro’) was profiled by ESI-HRMS at two snapshot time points: E-L 33 (grape berry still hard and green) and E-L 38 (grape berry at maturity). An untargeted analysis of the phloem sap enabled the identification of 476 metabolites, whereas the targeted analysis focused on eight main classes, namely acids, sugar and polyols, amino acids, phenolic acids, hormones, vitamins and N compounds, as well as on several minor classes like fatty acids, phenols or diols. Depending on the developmental stage of the vine, major differences were observed in the composition of cv. ‘Loureiro’ phloem sap in response to FD infection in terms of sugar, organic acid and amino acid content; furthermore, compounds involved in plant defense, such as salicylic acid, ABA, vitamin C and gallic acid also significantly changed. Overall, these results have contributed towards increasing knowledge about the complex interactions between vine and FD phytoplasma.