In this study, the yield of morphological parts, the saponin content and profile, and the antimicrobial and antioxidant activities of three Gypsophila species (G. paniculata, G. pacifica, and G. scorzonerifolia) were determined. Furthermore, an effort was made to develop guidelines on general principles of cultivation and recommendations with regard to raw material harvesting. Along with plant aging, a gradual increase in the size and weight of above- and underground parts of plants of three analysed Gypsophila species was noted. Profiling of GOTCAB (Glucuronide Oleanane-type Triterpenoid Carboxylic Acid 3,28-O-Bidesmosides) saponins was carried out with the use of liquid chromatography coupled with a quadrupole-orbitrap mass spectrometer and 10 major GOTCABs were tentatively identified in the Gypsophila roots. The main prosaponins of the Gypsophila saponins were gypsogenin 3-O-glucuronide and quillaic acid 3-O-glucuronide. The content of total saponins varied from 3.4 to 48.1 mg g−1 dry root weight, with the highest value in two- and three-year-old G. scorzonerifolia and G. pacifica roots. For production of raw material with high saponin content, G. scorzonerifolia and G. pacifica roots should be recommended for harvesting after at least three years of cultivation. It was also proved that the Gypsophila raw material was characterized by antimicrobial and antioxidant properties. The hexane extracts prepared from the roots of three Gypsophila species showed higher antifungal and antimicrobial activity than the methanol ones. Furthermore, simulated gastrointestinal digestion was applied to determine the polyphenol content and antioxidant potential of the in vitro bioaccessible fraction. Overall, G. pacifica was characterized by the highest polyphenol content and antioxidant activity, both in the case of undigested and digested samples. The in vitro digestion had no significant influence on the polyphenol content in all species. However, this process resulted in a decrease in Fe3+ reducing antioxidant power to 64.6% (G. scorzonerifolia) and DPPH• scavenging activity to 59.7% (G. scorzonerifolia). Conversely, it caused an increase up to 230.2% (G. scorzonerifolia) and 159.3% (G. pacifica) of ABTS•+ and OH• scavenging activities, respectively. Therefore, the roots of the examined Gypsophila species can be considered as a valuable source of phytochemicals, especially saponins, with antimicrobial and antioxidant properties. Our findings provide a basis for developing a valuable food additive or medicines as well as raw material for chemical and hygiene industries.