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Syringopicroside is a natural drug with antibacterial activity, which is the main ingredient of <i>Syringa oblata</i> Lindl (<i>S. oblata</i>). In order to further develop the application of <i>S. oblata</i> and evaluate the ability of syringopicroside against <i>Streptococcus suis</i> (<i>S. suis</i>), this investigation first applied an ultrasonic-assisted method to extract syringopicroside, and then response surface methodology (RSM) was performed to get the optimum condition. Based on RSM analysis, a second-order polynomial equation about the syringopicroside yield and four variables, including ultrasonic power, time, temperature, and liquid-to-solid ratio, was purposed. Through RSM prediction and model verification experiments, the optimum conditions were determined, as follows: ultrasonic time was 63 min, temperature was 60 °C, a liquid-to-solid ratio was set to 63 mL/g, and ultrasonic power was 835 W. Under this condition, a high syringopicroside yield was obtained (3.07 ± 0.13 mg/g), which was not significantly different with a predicated value. After separation and purification by HPD 500 microporous resin, then mass spectrum was applied to identify the main ingredient in aqueous extract. A minimal inhibitory concentration (MIC) assay revealed the value against <i>S. suis</i> of syringopicroside was 2.56 µg/µL and syringopicroside with sub-inhibitory concentrations that could effectively inhibit biofilm formation of <i>S. suis</i>. Besides, scanning electron microscopy analysis indicated syringopicroside could destroy the multi-layered aggregation structure of <i>S. suis</i>. Finally, molecular docking analysis confirmed that syringopicroside was combined with Orfy protein of <i>S. suis</i> through hydrogen bonds, hydrophobic interaction, and π-π stacking.