Murdannia pauciflora is an ethno-medicinally important plant; it was used for the first time to green- synthesize silver nanoparticles, with phytochemical analysis highlighting its ecofriendly approach and promising potential for diverse biomedical applications. Phytochemical screening revealed the presence of phenols, alkaloids, tannins, glycosides, flavonoids, and saponins. GC–MS analysis identified a total of 98 bioactive compounds in the extract. The synthesized AgNPs were characterized using UV–Visible spectroscopy, FTIR, XRD, SEM, EDX, zeta potential analysis, DLS, and TEM. XRD analysis confirmed the face-centered cubic (FCC) crystalline structure and rectangular morphology of the AgNPs, with an average particle size of approximately 12 nm and a surface charge of −30 mV, indicating good stability and uniform surface distribution. Cytotoxicity studies showed that the synthesized AgNPs exhibited an IC₅₀ value of 500 µg/mL against a pancreatic cancer cell line. Additionally, M. pauciflora AgNPs demonstrated significant α-glucosidase inhibitory activity with an IC₅₀ value of 78.44 µg/mL compared to the standard. The free radical scavenging assay revealed an IC₅₀ value of 120 mg/mL, indicating notable antioxidant activity. Overall, the green-synthesized M. pauciflora AgNPs exhibited good stability, uniform morphology, and promising antioxidant, antidiabetic, and cytotoxic activities, highlighting their potential applications in biomedical fields such as drug delivery, cancer therapeutics, and the development of antidiabetic formulations.