Background: Copper Silicate Nanoparticles (Cu-silicate NPs) have gained attention for their unique physical, chemical, and biological properties, making them promising candidates for antimicrobial and anticancer applications. Traditional chemical synthesis methods often involve hazardous substances, prompting the need for greener, more sustainable approaches. This study explores the synthesis of Cu-silicate NPs using Pseudomonas aeruginosa, a bacterium known for its metabolic capabilities, and evaluates their characterization, antimicrobial, and anticancer properties. Methods: Cu-silicate NPs were synthesized by incubating Pseudomonas aeruginosa with copper sulfate under controlled conditions. The resulting nanoparticles were purified and characterized using UV-Vis, TEM, XRD, DLS, and FTIR. Antimicrobial activity was assessed against bacterial pathogens, while anticancer activity was evaluated using cancer cell lines. Results: UV-Vis spectroscopy confirmed Cu-silicate NPs formation with a Surface Plasmon Resonance peak at 580 nm. TEM images revealed an average size of 35 ± 10 nm. XRD analysis indicated a Face-Centered Cubic (FCC) structure with characteristic peaks, while DLS measurements showed a dynamic diameter of 40 ± 5 nm and a zeta potential of -25 mV, indicating good stability. FTIR spectra identified functional groups associated with bacterial metabolites on the nanoparticle surface. The synthesized Cu-silicate NPs exhibited significant antimicrobial activity against various pathogens and demonstrated promising anticancer effects by inducing oxidative stress and apoptosis in cancer cell lines. Conclusion: The study successfully demonstrated a green synthesis approach for Cu-silicate NPs using Pseudomonas aeruginosa. The characterized nanoparticles showed potential for antimicrobial and anticancer applications, offering a sustainable alternative to conventional synthesis methods. Further research is needed to explore their full therapeutic potential and mechanisms of action.