Flavescence dorée phytoplasmas (FDp, 16SrV-C and -D) are plant pathogenic non-cultivable bacteria associated with a severe grapevine disease. The incidence of the two reference strains on cultivated grapevines is unbalanced, and mixed infections are rare. To investigate the interaction between the two strains, Catharanthus roseus plants were graft-infected with both strains, either simultaneously or sequentially. Different combinations of lateral and apical grafting were applied to avoid possible benefits due to graft position. The infection was monitored for four months through a new diagnostic protocol developed for differentiation and relative quantification of the two strains. Regardless of the temporal or spatial advantage at grafting, FD-C generally outcompeted FD-D. The prevalence of FD-C increased over time and, at the end of the experiment, FD-C was the unique strain detected in the aerial part and the roots of 74% and 90% of grafted plants, respectively. These data indicate that the interaction between the two strains results in competitive exclusion. Understanding the bases of the competition between FD-C and FD-D may contribute to explain the biology of the coexistence of different FDp strains under field conditions, aiming at identifying potential suppressor strains, which can provide alternative and environmentally sustainable solutions for FD control.