Based on antimicrobial circle diameter of Scutellaria baicalensis extract against Botryosphaeria dothidea, the extraction conditions of antibacterial active substances from Scutellaria baicalensis and the antibacterial stability of the active substances were determined. Experimental results show that when 80% ethanol is used as extraction solvent, the extract has strong bacteriostatic effect; the optimum extraction process condition includes ratio of material to liquid 1∶27.5, ultrasonic extraction time 2.5 h, extraction temperature 72.5 ℃, and ultrasonic power 850 W, under which the diameter of bacteriostasis circle reached 31.8 mm. The antimicrobial activity of 80% ethanol extract increased by 28.69%, compared to acetone extract in condition with ratio of material to liquid 1∶20, temperature 40 ℃, and 700 W ultrasound for 2.0 h. UV irradiation over 10 h , an alkaline environment, and the presence of metal ions Ag+, Fe3+, Cu2+ all showed inhibition effects on the activity of Scutellaria baicalensis extract. Compared to a pH 5 environment, the diameter of bacteriostasis circle decreased by 44.10% at pH 12. The activity of extracts was less affected by storage below 25 ℃ for about 40 days.