Antimicrobial-resistant bacterial disease has been recognized by WHO as a critical global health threat, Staphylococcus aureus posing particular concern due to its role in severe septic infections and bacteremia. This concern has led to an urgent need for alternative therapies, such as nanotechnology and plant-based approaches. The study focused on synthesizing plant-based silver nanoparticles from aqueous extract of Catharanthus roseus stem (CrS-AgNPs) prepared via the decoction process. The formation of CrS-AgNPs was confirmed by UV-Vis spectroscopy with a stable peak at 426 nm, followed by further characterization using FTIR and XRD analyses. The antimicrobial potential against pathogenic S. aureus was evaluated using the resazurin assay. Furthermore, SDS-PAGE was performed for whole cell protein profiling, and RT-PCR was used to analyze the expression of the virulence gene phospholipase C (plc). The results showed six functional peaks in the FTIR spectrum, while XRD analysis revealed a prominent peak at 38.33°, indicating a face-centered cubic geometry, and the calculated particle size is 26.74 nm. MIC and MBC were determined as 0.04 µg/mL and 0.32 µg/mL, respectively. While SDS-PAGE indicated protein-level alterations, RT-PCR showed no significant change in plc gene expression. Silver nanoparticles synthesized from C. roseus stem were successfully obtained, exhibiting functional groups of C≡N, H-C=O, and O-H, which contribute to their antimicrobial properties. Further investigation is needed to understand the underlying mechanisms and explore their therapeutic potential.