The pharmacodynamic substances in “Scrophulariae Radix–Fritillaria” and the molecular mechanisms underlying its therapeutic effects against goiter were analyzed through metabolomics and serum pharmaco-chemistry. A rat model of goiter was established using propylthiouracil (PTU), and the animals were treated using “Scrophulariae Radix–Fritillaria.” The efficacy of the drug pair was evaluated in terms of thyroid gland histopathology and blood biochemical indices. Serum and urine samples of the rats were analyzed by UPLC-Q-TOF/MS. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to screen potential biomarkers in urine and the corresponding metabolic pathways. The blood components of “Scrophulariae Radix–Fritillaria” were also identified, and their correlation with urine biomarkers was analyzed in order to screen for potential bioactive compounds. “Scrophulariae Radix–Fritillaria” mitigated injury to thyroid tissues and normalized the levels of the thyroid hormones FT3, FT4, and TSH. We also identified 22 urine biomarkers related to goiter, of which 19 were regulated by “Scrophulariae Radix–Fritillaria.” Moreover, urine biomarkers are involved in tryptophan metabolism, steroid hormone biosynthesis, and beta-alanine metabolism, and these pathways may be targeted by the drug pair. In addition, 47 compounds of “Scrophulariae Radix–Fritillaria” were detected by serum pharmacochemistry, of which nine components, namely, syringic acid, paeonol, cedrol, and cis-ferulic acid, fetisinine, aucubigenin, linolenic acid, ussuriedine, and 5-(methylsulfanyl)pentanenitrile, were identified as potential effective substances against goiter. To summarize, we characterized the chemical components and mechanisms of “Scrophulariae Radix–Fritillaria” involved in the treatment of goiter, and our findings provide an experimental basis for its clinical application.