In this study, gold nanoparticles (AuNPs) were synthesised biologically using cell-free extracts of the cyanobacterium Nostoc sp. strain HKAR-2 isolated from an Indian hot spring. UV-VIS spectroscopy, FTIR, X-ray diffraction, scanning electron microscopy (SEM), and transmission electron microscopy-selected area electron diffraction (TEM-SAED) were used to determine the morphological, structural, and optical properties of green synthesised AuNPs. The absorbance peak at 540 nm was observed due to the reduction of Au3+ to Au0 by cyanobacterial extract, indicating surface plasmon resonance (SPR) of the synthesised AuNPs. Characteristic In the XRD pattern, Bragg peaks at (111), (200), (220), and (311) facets of the face centre cubic (fcc) confirmed the crystalline nature of AuNPs. FTIR results revealed that proteins and amino acids play a role in the reduction of Au3+ to Au0 as well as the stability of AuNPs. A charge of -2.39Mv on the AuNPs was confirmed by zeta potential. The results of SEM and TEM confirmed the large agglomerated shape of AuNPs with sizes ranging from 10-100 nm. Their antibacterial, antifungal, and anticancer properties were also investigated in vitro against plant bacterial strains, fungal strains, and MCF-7 cells. AuNPs also demonstrated dose-dependent cytotoxic activity against MCF-7 human breast cancer cells, with an IC50 of 250 g/mL.