The genusScrophulariais one of the largest genera belonging to the Scrophulariaceae family. Different members of the genus exhibit an interesting, wide spectrum of bioactivities. Accordingly, the current study aimed to investigate, for the first time, the chemical composition of the essential oil ofScrophularia peyroniiPost. from Jordan. Additionally, extracts obtained from the aerial parts with solvents of different polarities were assayed for their phytochemical constituents and in vitro antioxidant activities. The major constituents detected in the essential oil, as revealed by GC/MS analysis, contained mainlyZ,Z-farnesyl acetone (11.04%), β-elemene (6.36%),n-octanal (5.98%), and spathulenol (4.58%). Each of the aqueous methanol (Sp-M) and butanol (Sp-B) extracts contained flavonoids, saponins, anthraquinone, and glycosides. Both extracts were evaluated for their total phenolic content (TPC), total flavonoid content (TFC), and their in vitro antioxidant activity, which were assayed using the DPPH radical scavenging activity and ABTS radical scavenging methods. Additionally, the two extracts were then subjected to LC-ESI-MS/MS for the qualitative determination of their secondary metabolite content, especially in flavonoids and phenolic compounds. The results showed that the Sp-B extract ofS. peyroniihad the highest contents of both phenolic compounds and flavonoids and showed high radical scavenging activity, as determined by the two assay methods, when compared with the Sp-M extract. The LC-ESI-MS/MS analysis resulted in the detection of 21 compounds, including 8 flavonoids, 6 phenolic acids, 6 iridoids, and 2 acids. Although the majority of compounds were detected in both extracts, it was noticed that scropolioside B, 6′-O-cinnamoylharpagide, isoferulic acid, and 6-O-methylcatapol were only detected in the Sp-M fraction.