In the present study, biosynthesis of gold nanoparticles (AuNPs) by the cells (cells-AuNPs) and cell-free extracts (extracts-AuNPs) of a new fungus Mariannaea sp. HJ was reported. The as-synthesized particles were characterized by UV-vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The effects of different parameters on AuNP biosynthesis were investigated, and initial gold ion concentration of 2 mM, pH 7, was demonstrated to be suitable for both cells-AuNP and extracts-AuNP syntheses. The cells-AuNPs were of various shapes, including sphere, hexagon, and irregular shapes, with an average size of 37.4 nm, while the extracts-AuNPs were almost spherical and pseudo-spherical with an average size of 11.7 nm. XRD pattern suggested that the crystal structure of both AuNPs was face-centered cubic. FTIR spectra implied that some biomolecules from the fungal cell walls or cell-free extracts were involved in the formation of AuNPs. The as-synthesized AuNPs were demonstrated to have excellent catalytic activities for the reduction of 4-nitrophenol with the catalytic rate constants of 5.7 × 10/s for cells-AuNPs and 24.7 × 10/s for extracts-AuNPs. To the best of our knowledge, this is the first report on AuNP biosynthesis by Mariannaea sp.