This work aimed to interrogate the role that the starting graphitic material played on the physicochemical properties of graphene oxide (GO) sheets and their impact on mammalian cell viability following exposure to those flakes. Three different GO thin sheets were synthesised from three starting graphite material: flakes (GO-f), ground (GO-g) and powder (GO-p) using a modified Hummers’ method. The synthetic yield of this methodology was found to differ according to type of starting material, with GO-p resulting in most efficient yields. Structural and morphological comparison of the three GO sheet types were carried out using transmission electron microscopy (TEM) and atomic force microscopy (AFM). Optical properties were measured using UV/Visible and fluorescence spectroscopy. Surface characteristics and chemistry were determined using a battery of techniques. Exposure to human cells was studied using the human A549 lung epithelial cultures. Our results revealed that all three GO samples were composed of few-layer sheets with similar physicochemical and surface characteristics. However, significant differences were observed in terms of their lateral dimensions with GO-p, prepared from graphite powder, being the largest among the GOs. No cytotoxicity was detected for any of the GO samples following exposure onto A549 cells up to 48h. In conclusion, the form and type of the starting graphite material is shown to be an important factor that can determine the synthetic yield and the structural characteristics of the resulting GO sheets.