Marine microorganisms associated with macroalgae have currently been identified as an environmentally safe and efficient technique to use as biofactories in order to produce metal nanoparticles. The current investigation focuses to identify the capability of marine bacteria Exiguobacterium aestuarii ADCG SIST 3 for the extracellular synthesis of gold nanoparticles (AuNPs). The AuNPs that are synthesized were characterized by different techniques and analyzed for their applications in anticancer activity on normal and cancer cells. To the best of our knowledge, this study is first to systematically examine extracellular synthesis of AuNPs by the halotolerant bacteria Exiguobacterium aestuarii ADCG SIST 3 associated with seaweed. The characterization of synthesized AuNPs were carried out using Fourier Transform- Infrared Spectroscopy (FTIR), Raman Spectroscopy, Field Emission Scanning Electron Microscope (FE-SEM), High-Resolution Transmission Electron Microscope (HR-TEM), Particle size analyzer (DLS), Zetasizer, and UV–vis Spectrophotometer. With an average size of 37 nm, the fabricated AuNPs were observed to have a spherical-like morphology. The cytotoxic response of synthesized AuNPs was assessed by MTT assay on Vero and MCF-7 cell lines. The antioxidant potential of the synthesized AuNPs and apoptosis studies were also carried out which resulted in excellent activity in the MCF-7 cells with potential application in the medical field.