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Over past decades, the green method of synthesizing metal nanoparticles has acquired more attentiveness by scientific consensus because of its industrial and biomedical applications. This study focuses on the anti-proliferative effectiveness of AgNPs synthesized from Rotheca serrata (L.) Steane & Mabb. flower bud extract against the PANC-1 cell line in vitro. Various analytical instruments were utilized to visualize the formation of RsFb-AgNPs, such as UV-Vis spectroscopy, FT-IR, SEM, EDS, TEM, XRD, Zeta potential, and DLS analysis. The biosynthesis of RsFb-AgNPs was observed by a change in color and UV-Vis spectroscopy (415 nm). The FT-IR spectra exhibited the existence of many functional groups. XRD confirmed the crystallinity of the AgNPs. Morphology and elemental mapping were assessed by SEM and EDS analysis. The TEM micrograph revealed spherical-shaped particles with sizes ranging from 12 to 40 nm. Zeta potential and DLS analysis were used to measure surface charge and particle size. Biological properties, including the antioxidant, antimicrobial, and anticancer properties of synthesized RsFb-AgNPs, exhibited dose-dependent activities. In DPPH assay, synthesized RsFb-AgNPs inhibited the scavenging of free radicals in a dose-dependent manner. In addition, the resultant RsFb-AgNPs displayed moderate antimicrobial activity against tested pathogens. Further, the anti-proliferative efficacy of biosynthesized RsFb-AgNPs was determined against the PANC-1 cell line using the MTT assay. The results revealed a dose-dependent decrease in viability of cancer cells with an IC50 value of 36.01 µg/mL. Flow cytometry was then used to confirm the apoptotic effects by double staining with annexin V/PI. In response to the pancreatic ductal adenocarinoma cell line, the results showed notable early and late apoptosis cell population percentages. In conclusion, the synthesized RsFb-AgNPs revealed a potential anticancer agent that can induce apoptosis in the PANC-1 cells. [ABSTRACT FROM AUTHOR]