In the present study, we report cytotoxic and antimicrobial potential of gold nanoparticles (Au NPs) synthesized using chitosan derived from squilla shell wastes. Here we adopted ecofriendly approach for the synthesis of Au NPs and characterized them using UV-visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, atomic force microscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS) methods. The UV-visible spectroscopic analysis revealed an absorption peak at 529 nm, which represents the Surface Plasmon Resonance of Au NPs. TEM analysis showed that the biosynthesized Au NPs were spherical in shape with an average size of 80 to 82 nm. Interestingly, the biosynthesized Au NPs showed antimicrobial activity against selected Gram-positive and negative bacterial isolates and also showed activity against fungal pathogens. Further, the cytotoxic effect of the synthesized Au NPs against MCF-7 cell lines was assessed by MTT assay with IC50 value of 250 μg mL. In addition, double staining of treated MCF-7 cells with acridine orange and ethidium bromide shows that the Au NPs exert apoptosis mediated cytotoxicity.