This study highlights a novel, eco-friendly approach for synthesizing silver nanoparticles (AgNPs) using the Pandanus dubius extract. This study characterized the resulting Pds-AgNPs and evaluated their antioxidant, antibacterial, and anticancer properties. The characterization of Pds-AgNPs used various methods UV-Vis spectroscopy confirmed the absorption peak at 458 nm, while FT-IR analysis identified the functional groups responsible for reduction and stabilization. X-ray diffraction (XRD) analysis provided insights into the crystallographic features of the nanoparticles, and scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealed their spherical shape. The biomedical potential of Pds-AgNPs was thoroughly investigated. Antioxidant activity, significant free radical scavenging capabilities, with the Pds-AgNPs outperforming the methanol extract. The antibacterial activity of Pds-AgNPs was tested against human pathogens, and the Pds-AgNPs exhibited strong antibacterial efficacy, surpassing the performance of the methanol extract alone. Furthermore, the anticancer properties of Pds-AgNPs were assessed using in vitro cytotoxicity assays on A549 human lung cancer cells. The IC<inf>50</inf> values of 40.84 ± 0.092 µg/mL for the Pds-AgNPs and 54.88 ± 0.105 µg/mL for the extract, highlighting the enhanced therapeutic potential of the nanoparticles (NPs). These findings underscore the potential of green-synthesized Pds-AgNPs as multifunctional therapeutic agents with robust antioxidant, antimicrobial, and anticancer properties. This study is the first to report the analysis of phytochemicals and the sustainable, eco-friendly synthesis of Pds-AgNPs using a P. dubius plant-mediated approach. These findings provide a basis for further research on the use of Pds-AgNPs in biomedical applications. © The Author(s) 2025.