Gold nanoparticles (Au-NPs) have several uses for nanobiotechnologists because of their beneficial biomedical properties. Alginates have various biomedical and industrial applications. The aim of this study is to extract alginate from Azotobacter chroococcum, synthesize chemical Au-NPs (Ch/Au-NPs), and load the NPs with the extracted alginate to form Azotobacter alginate gold nanocomposites (Azto/Alg-Au-NCMs). The Ch/Au-NPs and Azto/Alg-Au-NCMs were characterized by UV-spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy, energy-dispersive spectroscopy (EDS), X-ray diffraction (XRD), thermogravimetric analysis (TGA), zeta potential, and transmission electron microscopy (TEM). The anticancer activities were determined using the breast cancer cell line MCF-7, human lung cancer cell line H1299, and Vero cell line. The results obtained by UV-spectroscopy exhibited a surface plasmon resonance that was clearly noticeable at 530 nm, and the EDS analysis proved that gold was present in percentages of 50.11 and 28.08 in the Ch/Au-NPs and Azto/Alg-Au-NCMs, respectively. There were several similarities between the alginic acid and the alginate extracted from A. chroococcum, and small modifications were proved by FT-IR spectroscopy. Negative charges were shown by the zeta potential. Crystalline and cubic NPs were shown by XRD analysis and TEM. TGA demonstrated the purity of the Ch/Au-NPs and the existence of organic compounds in the Azto/Alg-Au-NCMs. Both the Ch/Au-NPs and Azto/Alg-Au-NCMs had antibacterial activities against Staphylococcus aureus ATCC 25923, Proteus mirabilis, Enterobacter sp., and Pseudomonas aeruginosa and possessed anticancer activities against MCF-7 and H1299.