The current study fabricated a nanocomposite based on gold nanoparticles and carboxy methyl cellulose (CMC-AuNPs). The prepared nanocomposite (CMC-AuNPs) was fully characterized using FTIR, EDX, SEM, XRD, mapping, zeta potential, DLS, and TEM. In the colloidal dispersion, spherical and dispersed CMC-AuNPs with a diameter range of 10–90 nm were found. CMC-AuNPs displayed modest antibacterial activity against Staphylococcus aureus and Bacillus cereus, with minimal inhibitory concentrations (MICs) value of 25 μg/ml, while CMC-AuNPs had higher antibacterial activity against Klebsiella oxytoca and Escherichia coli with MICs value 50 and 100 μg/ml respectively. In addition, CMC-AuNPs displayed antifungal activities against C. albicans, A. terreus, A. niger, and A. fumigatus with inhibitory zones 20, 13, 23, and 26 mm respectively at 500 μg/ml. The MCF-7 recorded the lowest IC50 values (2.56 ± 0.19 μg/mL) for CMC-AuNPs, suggesting a strong cytotoxic effect on breast cancer cells. The percentage of total apoptosis was in CMC-AuNPs treated cells (38.81 ± 1.99%) compared to 0.61 ± 0.022% for control. As a result of our findings, we believe that the anticancer function of these CMC-AuNPs is attributable to the activation of necrosis and apoptosis in breast cancer cells via enhanced caspase-8 and -9 activity and decreased VEGFR-2 levels. In conclusion, CMC-AuNPs were successfully synthesized and exhibited antibacterial, antifungal, and anticancer activities, which can be used in the pharmaceutical and medical fields.