Secondary metabolites derived from plants have demonstrated significant biological activity and hold both edible and medicinal significance. These compounds play a crucial role in the development of pharmaceuticals, particularly in the context of inflammatory disorders and chronic diseases associated with oxidative stress. The objective of this study was to investigate the chemical characterization, antioxidant potential, and antibacterial properties of the aqueous extract of Anacyclus pyrethrum (AEAP). To achieve this, we employed various analytical techniques including HPLC–ESI–MS/MS, Fourier transform infrared, X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. The antioxidant activity of the AEAP was assessed using DPPH (2,2-Diphenyl-1-picrylhydrazyl) and reducing power assays, while antibacterial activity was evaluated against both Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecium) using the broth microdilution assay. Our findings demonstrated significant antioxidant activity of the AEAP, as well as broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. HPLC analysis identified important bioactive compounds, notably pellitorine and 3,4-dihydroxybenzoic acid, known for their antioxidant and antibacterial properties. Overall, the AEAP demonstrated potent antioxidant and antibacterial activities, suggesting its potential as a valuable natural source of bioactive compounds with various therapeutic applications.