Myco-synthesis of stable gold nanoparticles (GNP) using Aspergillus flavus filtrate was carried out. Myco-synthesized GNP were characterized by UV-Vis spectroscopy, high resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). GNP showed pinkish violet colour with a peak at about 530-540 nm as detected by UV-Vis spectroscopy. HRTEM showed spherical, hexagonal, rectangular and anisotropic crystalline nanoparticles with 10-50 nm in size. The size was confirmed by DLS and it was 39.5 ± 16 nm. FTIR analyses of synthesized GNP showed the presence of amide and carbonyl functional groups which indicates the presence of peptides that are involved in the synthesis and stability of GNP. Nitrate reductase activity was found to be 0.83 µmole/min/ml of filtrate. The optimum conditions for the synthesis of GNP were filtrate concentration of 10%, 1 mM HAuCl₄ in aqueous solution, 0.1% Tween 20, incubation period of 30 min, and an incubation temperature of 30 °C under static and illumination conditions. GNP synthesized using A. flavus filtrate showed a potent cytotoxic effect on both breast and lung carcinoma human cell lines (LC₅₀ was 8 and 80 ng/ml, respectively). However, they were non-toxic to normal human cells at the same tested concentrations. This study used a simple, clean, quick and inexpensive approach for the synthesis of GNP. The synthesized GNP showed anticancer activity and they can be used in biomedical applications.