The development of multifunctional nanomaterials with enhanced biocompatible potential is crucial for effective biomedical applications. Herein we propose electrospun silk fibroin/cellulose acetate/gold-silver nanoparticles (CA/SF/Au-Ag) composite nanofiber for anticancer applications. The silk fibroin and cellulose acetate serving as the reducing and stabilizing agent for Ag+ and Au+ ions with improved biocompatibility. The fabricated CA/SF/Au-Ag nanofiber was studied with different functional, surface and crystallographic techniques. The CA/SF polymer matrix was formed in the needle and rod-shaped morphology with the range of 86.02 ± 57.35 nm in diameter and the Au and Ag NPs were embedded on the fiber matrix with an average size of 17.32 nm and 53.21 nm respectively. Further, it strongly triggers the cytotoxic effects against MCF-7 and MDA-MB-231 human breast cancer cells with an effective IC50 value. Our findings implied that CA/SF/Au-Ag composite nanofibers are an effective material for safer anticancer applications.