The cell free extracts of Candida parapsilosis ATCC 7330 are more efficient than the whole resting cells of the yeast in the synthesis of directly usable gold nanoparticles as revealed by this systematic study. Cell free extracts yielded gold nanoparticles of hydrodynamic diameter (50–200 nm). In this study, the total protein concentration influences the nanofabrication and not only the reductase enzymes as originally thought. Powder X-ray diffraction studies confirm the crystalline nature of the gold nanoparticles. Fourier Transform Infra Red spectroscopy and thermal gravimetric analysis suggests that the biosynthesized gold nanoparticles are capped by peptides/proteins. Dispersion experiments indicate a stable dispersion of gold nanoparticles in pH 12 solutions which is also confirmed by electron microscopic analysis and validated using a surface plasmon resonance assay. The effectiveness of the dispersed nanoparticles for the reduction of 4-nitrophenol using sodium borohydride as a reductant further confirms the formation of functional gold nanoparticles. It is also reported that gold nanoparticles with mean particle diameter of 27 nm are biosynthesized inside the whole cell by transmission electron microscopy analysis. With optimized reaction conditions, maximum gold bioaccumulation with the 24 h culture age of the yeast with cellular uptake of ~1010 gold atoms at the single cell level is achieved but it is not easy to extract the gold nanoparticles from the whole resting cells. © 2016, The Author(s).