The anti-phytoplasma activities of surfactin (derived from Iranian native Bacillus subtilis isolates) and tetracycline towards Candidatus "Phytoplasma aurantifolia", the agent of lime Witches' broom disease, were investigated. HPLC was used to quantify the surfactin production in four previously characterized native surfactin-producing strains, and the one producing the highest amount of surfactin (about 1,500 mg/l) was selected and cultivated following optimized production and extraction protocols. Different combinations of purified surfactin and commercial tetracycline were injected into artificially phytoplasmainfected Mexican lime seedlings using a syringe injection system. An absolute quantitative real-time PCR system was developed to monitor the phytoplasma population shifts in the lime phloem during 3 months following the injections. The results revealed that the injections of surfactin or tetracycline had a significant inhibitory effect on Candidatus "P. aurantifolia". However, the combined treatment with both surfactin and tetracycline (1:1) resulted in the highest inhibition due to a synergic effect, which suppressed the phytoplasma population from about 2×10(5) to less than 10 phytoplasma units/g plant tissue.