In this study, silver nanoparticles (AgNPs) were synthesized by a microwave-assisted green chemistry method using a crude extract of Macrolepiota procera mushroom. The synthesized M. procera-AgNPs (MP-AgNPs) were characterized by UV-VIS spectroscopy, fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), dynamic light scattering (DLS) technique, and scanning transmission electron microscopy (S-TEM). The cytotoxic activity of synthesized MP-AgNPs was investigated in human breast cancer (MCF-7), lung cancer (A549), osteosarcoma (Saos-2), and colon cancer (HT-29) cell lines. HSP27, HSP70, and HSP90 inhibitory potential of MP-AgNPs were determined in cancer cells by RT-PCR and Western blotting experiments. To analyze the apoptotic profile of MP-AgNPs in cancer cell lines, the expression levels of apoptotic and anti-apoptotic genes were determined using RT-PCR. The results showed that MP-AgNPs were exhibited anticancer activity in MCF-7, A549, Saos-2, and HT-29 cell lines. MP-AgNPs decreased gene and protein expression levels of HSP27, HSP70, and HSP90 in cancer cells. Also, MP-AgNPs induced apoptotic cell death mechanisms in MCF-7, A549, Saos-2, and HT-29 cell lines. MP-AgNPs are found to be significant metallic nanoparticle systems as efficient HSP inhibitors in the treatment of cancer.