The transmission of microbial pathogens by insect vectors can be affected by the insect’s microbial symbionts, which may compete in colonizing organs, express antagonistic factors or activate host immune response. Acetic acid bacteria of the genus Asaia are symbionts of the leafhopper Scaphoideus titanus, which transmits Flavescence dorée phytoplasma. These bacteria could be used as control agents against the disease. Here, we experimentally investigated the interaction between different strains of Asaia and phytoplasma transmission in the laboratory by using the model leafhopper Euscelidius variegatus and the plant host Vicia faba. We found that uncultivable and low concentrations of Asaia phylotypes were associated with E. variegatus. When we supplied different Asaia strains isolated from other insects and exhibiting different phenotypes to E. variegatus orally, the bacteria stably colonized the leafhopper, reached relatively higher densities and could then be isolated from the host. We conducted transmission trials of Flavescence dorée phytoplasma with individuals colonized with three exogenous Asaia strains. When the phytoplasma became established in the bodies of E. variegatus, leafhoppers were able to transmit it to broad beans, with transmission rates ranging from 33 to 76% in different experiments. However, leafhoppers that were colonized by one of the Asaia strains producing an air–liquid interface biofilm exhibited significantly reduced phytoplasma acquisition, with infection rates at 5–28%, whereas they were 25–77% in control insects. Although the mechanisms regulating this interference remain to be elucidated, our results provide evidence of the potential use of Asaia as a biocontrol agent.