Baicalin and wogonoside are two of the most abundant flavonoid glycosides in the root of Scutellaria baicalensis Georgi, which is a widely used peroral herbal medicine with anticancer, antiviral, antibacterial and anti-inflammatory properties. In the present study, the effects of intestinal microecology on the metabolism and pharmacokinetics of orally administered baicalin and wogonoside were investigated by UPLC-QTOF/MS measurement of the difference in metabolites between normal and antibiotic-pretreated rats. In the antibiotic-pretreated rats, the plasma concentration-time profile and pharmacokinetic parameters of the two flavonoid glycosides and their relevant aglycone forms were significantly changed compared with those in normal rats. Further, hydrolysis and glucuronidated metabolites were not detected in the cecum contents and urine samples from antibiotic-pretreated rats. These results suggested that intestinal microbiota may play a key role in the pharmacokinetics and metabolism of peroral baicalin and wogonoside. According to our findings, it is recommended that the root of S. baicalensis should not be co-administered with antibiotics in clinical use.