The study of Ammoides pusilla, a Tunisian medicinal plant, explored its chemical composition and biological activities, highlighting its under-exploited therapeutic potential. The essential oil, obtained by steam distillation, reveals twenty major compounds, including perilic aldehyde, β-phellandrene, and o-cymene. Two new natural constituents were identified in the cyclohexane extract and four in the dichloromethane extract. DPPH and ABTS tests showed that methanol extract exhibited the highest antioxidant activity, giving values of 78.9% and 65.5%, respectively, at 50 µg/mL. Its anti-diabetic activity (IC50 = 25.0 µg/mL) exceeds that of acarbose. The anti-SOD activity of methanol extract also showed promise, at 73.3% at 50 µg/mL. Essential oil and ethyl acetate extract showed notable inhibition of xanthine oxidase activity, reaching 69.0%. In addition, the essential oil demonstrated strong anti-AChE (63.23% at 50 µg/mL) and anti-inflammatory (IC50 = 31.0 µg/mL) activity. In terms of cytotoxicity, the methanol extract was effective against the HCT116 cell line (IC50 = 20.9 µg/mL), and all extracts showed activity against MCF7, OVCAR-3, and IGROV-1 cells, with IC50 values ranging from 4.0 to 25.0 µg/mL. This result underlines the potential of Ammoides pusilla extracts as important sources of bioactive compounds for therapeutic applications. Further research is needed to fully exploit these activities in drug development. [ABSTRACT FROM AUTHOR]