Salvia hispanica L. is an annual herbaceous plant commonly known as “Chia”. It has been recommended for therapeutic use because of its use as an excellent source of fatty acids, protein, dietary fibers, antioxidants, and omega-3 fatty acids. A literature survey concerning phytochemical and biological investigations of chia extracts revealed less attention towards the non-polar extracts of S. hispanica L. aerial parts, which motivates us to investigate their phytochemical constituents and biological potentials. The phytochemical investigation of the non-polar fractions of S. hispanica L. aerial parts resulted in the tentative identification of 42 compounds using UPLC-ESI-MS/MS analysis with the isolation of β-sitosterol (1), betulinic acid (2), oleanolic acid (3), and β-sitosterol3-O-β-D-glucoside (4). GLC-MS analysis of the seeds’ oil showed a high concentration of omega-3 fatty acid, with a percentage of 35.64% of the total fatty acid content in the seed oil. The biological results revealed that the dichloromethane fraction showed promising DPPH radical-scavenging activity (IC50 = 14.73 µg/mL), antidiabetic activity with significant inhibition of the α-amylase enzyme (IC50 673.25 µg/mL), and anti-inflammatory activity using in vitro histamine release assay (IC50 61.8 µg/mL). Furthermore, the dichloromethane fraction revealed moderate cytotoxic activity against human lung cancer cell line (A-549), human prostate carcinoma (PC-3), and colon carcinoma (HCT-116) with IC50s 35.9 ± 2.1 µg/mL, 42.4 ± 2.3 µg/mL, and 47.5 ± 1.3 µg/mL, respectively, and antiobesity activity with IC50 59.3 µg/mL, using pancreatic lipase inhibitory assay. In conclusion, this study’s findings not only shed light on the phytochemical constituents and biological activities of the non-polar fractions of chia but also should be taken as a basis for the future in vivo and clinical studies on the safety and efficacy of chia and its extracts. Further study should be focused towards the isolation of the active principles of the dichloromethane fraction and studying their efficacy, exact mechanism(s), and safety, which could benefit the pharmaceutical industry and folk medicine practitioners who use this plant to cure diseases.