A comprehensive metabolite profiling of the medicinal plant Silene viridiflora using an UHPLC-ESI-MS/MS method is described for the first time. A total of 71 compounds were identified and annotated, the most common of which were flavonoids, triterpene glycosides, and ecdysteroids. The three major compounds schaftoside, 26-hydroxyecdysone, and silviridoside can be chosen as the markers for the assessment of the quality of S. viridiflora preparations. The methanol extract and a variety of metabolites identified in S. viridiflora were screened for their cytotoxic and Wnt pathway-inhibiting activities against triple-negative breast cancer (TNBC), the deadliest form of cancer in women. 2-Deoxy-20-hydroxyecdysone with submicromolar IC50 was identified as a result. The structure–activity relationship derived from the data from the in vitro proliferation assay showed that the hydroxyl group present at position C-2 of steroid core reduces the ecdysteroids’ cytotoxicity against cancer cells.