Despite decades of research, breast cancer therapy remains a great challenge. Docetaxel is an antimicrotubule agent that is effectively used for the treatment of breast cancer. However, its clinical use is significantly hampered by its low water solubility and systemic toxicity. The current study was designed to prepare docetaxel (DXL)-loaded silk-fibroin-based nanoparticles (SF-NPs) and to screen their potential antitumor activity against breast cancer cell lines. DXL-loaded SF-NPs were prepared using a nanoprecipitation technique and were evaluated for particle size, zeta potential, entrapment efficiency, and in vitro release profile. In addition, DXL-loaded SF-NPs were screened for in vitro cytotoxicity, cellular uptake, and apoptotic potential against MCF-7 and MDA-MB-231 breast cancer cell lines. The prepared DXL-loaded SF-NPs were 178 to 198 nm in diameter with a net negative surface charge and entrapment efficiency ranging from 56% to 72%. In vitro release studies exhibited a biphasic release profile of DXL from SF-NPs with sustained drug release for 72 h. In vitro cell studies revealed that entrapment of DXL within SF-NPs significantly improved cytotoxic potential against breast cancer cell lines, compared to the free drug, and enhanced cellular uptake of DXL by breast cancer cells. Furthermore, the accumulation in the G2/M phase was significantly higher in cells treated with DXL-loaded SF-NPs than in cells treated with free DXL. Collectively, the superior antitumor activities of DXL-loaded SF-NPs against breast cancer cells, compared to free DXL, could be ascribed to improved apoptosis and cell cycle arrest. Our results highlighted the feasibility of using silk fibroin nanoparticles as a nontoxic biocompatible delivery vehicle for enhanced therapeutic outcomes in breast cancer.