Synthesis of silver nanoparticles using the leaf extract of Melaleuca alternifolia (M-AgNPs), as evidenced by various physicochemical characterizations, including UV-Visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDAX), high-resolution transmission electron microscopy (HR-TEM), and zeta potential analysis. The UV-Vis spectrum exhibited a characteristic absorption peak at 402 nm, confirming the formation of M-AgNPs. FT-IR analysis identified the presence of various functional groups associated with the silver nanoparticles. DLS measurements indicated a hydrodynamic size of 45.79 nm with a polydispersity index (PDI) of 0.335. Zeta potential analysis revealed a value of -21 mV, suggesting good stability of the nanoparticles. XRD analysis showed a crystalline size of 25.47 nm and confirmed the face-centered cubic (FCC) structure. SEM images revealed well-defined and uniformly dispersed nanoparticles. EDAX confirmed the presence of silver at the elemental level. HR-TEM analysis demonstrated that the actual size of the nanoparticles was approximately 10 nm. Antimicrobial studies demonstrated the effectiveness of the synthesized nanoparticles against both bacterial and fungal strains. The antioxidant activity of the M-AgNPs was measured at 41.17 µL/mL. Furthermore, cytotoxicity studies showed that the nanoparticles exhibited an IC₅₀ value of 8.16 µg/mL against MCF-7 breast cancer cells. The synthesized M-AgNPs possess significant potential as therapeutic agents, particularly against MCF-7 cancer cells, and may serve as promising candidates for future medical applications.