Abstract: In the present study, the effect of surfactin derived from Iranian native Bacillus subtilisisolates has been investigated against phytoplasma Candidatus “Phytoplasma aurantifolia” agentof lime witches broom disease (WBDL) which causes a considerable loss in lime production inIran and other countries in the region of Persian Gulf. For this purpose, eight surfactin producingisolates were evaluated for quantity of surfactin production by HPLC. Quantitative analysis byHPLC showed that one strain had the highest surfactin production (650mg/l) and were selectedfor surfactin extraction. After optimization of surfactin extraction, it was injected into witchesbroom phytoplasma-infected seedlings of lime by using syringe injection. To comparetetracycline effect with surfactin effect against phytoplasma activity and also to investigateprobable synergistic effect of them, we planned to also treat plants with both mentionedantibiotics at the same time. Two specific primers pairs were designed for qualitative andquantitative detection of phytoplasma in infected plants. Real Time PCR conjugated withFluorescent SYBR® Green I dye and absolute quantification has been developed for rapid,sensitive and quantitative analysis to assess surfactin bioactivity against phytoplasma, and also todetermine concentration of phytoplasma in infected seedlings before and after treatment. Theresults showed that the combination of surfactin had caused significant reduction of phytoplasmapopulation in infected plants, and also the combination of surfactin and tetracycline had thehighest toxicity against phytoplasma.