Scutellaria barbataD. Don (SB, Chinese: Ban Zhi Lian), a well-known medicinal plant used in traditional Chinese medicine, is rich in flavonoids. It possesses antitumor, anti-inflammatory, and antiviral activities. In this study, we evaluated the inhibitory activities ofSBextracts and its active components against HIV-1 protease (HIV-1 PR) and SARS-CoV2 viral cathepsin L protease (Cat L PR). UPLC/HRMS was used to identify and quantify the major active flavonoids in differentSBextracts, and fluorescence resonance energy transfer (FRET) assays were used to determine HIV-1 PR and Cat L PR inhibitions and identify structure–activity relationships. Molecular docking was also performed, to explore the diversification in bonding patterns of the active flavonoids upon binding to the two PRs. ThreeSBextracts (SBW, SB30, and SB60) and nine flavonoids inhibited HIV-1 PR with an IC50range from 0.006 to 0.83 mg/mL. Six of the flavonoids showed 10~37.6% inhibition of Cat L PR at a concentration of 0.1 mg/mL. The results showed that the introduction of the 4′-hydroxyl and 6-hydroxyl/methoxy groups was essential in the 5,6,7-trihydroxyl and 5,7,4′-trihydroxyl flavones, respectively, to enhance their dual anti-PR activities. Hence, the 5,6,7,4′-tetrahydroxyl flavone scutellarein (HIV-1 PR, IC50= 0.068 mg/mL; Cat L PR, IC50= 0.43 mg/mL) may serve as a lead compound to develop more effective dual protease inhibitors. The 5,7,3′,4′-tetrahydroxyl flavone luteolin also showed a potent and selective inhibition of HIV-1 PR (IC50= 0.039 mg/mL).