Abstract: The synthesis of nanoparticles from biological sources has gained significant attention recently, however, with much emphasis on the novelty of sources. This study aims to provide a comprehensive overview on the aspects of fungal age and type of cultivation on the properties of the nanoparticles. The effect of age of Fusarium oxysporum developed in submerged culture and on solid agar medium was evaluated. When the gold salt solution was added to the fungal colony on agar medium, a deep purple colored radial zone developed in the 3 days old mycelium. The nanoparticles in the radial purple zone had two distinct characteristics when the agar blocks were dried by either lyophilization or heating in a hot air oven. The lyophilized sample contained square bi-pyramidal particles (1 to 2 μm). The oven-dried sample contained abundant truncated triangular nanoplates (150 to 500 nm), decahedrons and icosahedrons (20 to 70 nm), and a few triangular and hexagonal nanoplates (50 nm). The mycelium when suspended in 1 mM HAuCl4·3H2O presented distinct geometries of the nanoparticles in the cytoplasm and periplasm. The formation of gold nanoparticles of different morphologies within the cytoplasm, and the periplasm has implications in Surface Enhanced Raman Spectroscopic (SERS) measurements. Graphic Abstract: HRTEM images of hyphae of Fusarium oxysporum suspended in 1 mM HAuCl4·3H2O for 24 h. The nanoparticles present in the electron dense cytoplasm are markedly different from those in the periplasmic space.[Figure not available: see fulltext.]. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.