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This paper reveals a novel and greener strategy to biosynthesize Cobalt-Ferrite-Silver trimetallic nanoparticles (Co-Fe-AgNPs) using the Citrus limon extract as a bio-reducing agent. Depending on the Transmission and Scanning Electron Microscopy (TEM and SEM), the prepared nanocomposites had a relatively small size with an average of 20 nm with a spherical shape. Moreover, The Energy Dispersive X-ray (EDX) showed the distribution of elements within the nanoparticles with various concentrations of C, K, S, O, Fe, Co and Ag. Finally, the potential ability of nanoparticles to combat the bacteria and biofilm formation was studied in-vitro. For example, a 125 µM nanocomposite is needed to kill Salmonella, and 62.5 µM inhibits its growth. Moreover, 30 µM is enough to inhibit biofilm formation. Furthermore, the anticancer activity was evaluated against MCF7 and HEPG2 cell lines with IC50 of 35.5 and 43.5 μg/mL, respectively. The findings suggest that Co-Fe-AgNPs could be a potential antibacterial and anticancer agent.