Multi-hole injectors for direct-injection spark-ignition engines offer atomisation benefits and flexibility in fuel targeting by selection of the number and orientation of the nozzle's holes. However, the spray formation process from multi-hole injectors has been mostly studied for hot fuel conditions and no quantitative data really exist with fuel temperatures representative of engine operation at cold-start. The challenge is further complicated by the predicted fuel stock which will include a significant bio-derived component presenting the requirement to manage fuel sustainability. The thermophysical properties of bio-components like ethanol differ markedly from typical hydrocarbons found in gasoline like iso-octane. Moreover, the production of anhydrous ethanol fuel involves separating water and ethanol by way of distillation and dehydration in an energy intensive process. This work presents results from an optical investigation into the effect of atomisation for iso-octane, anhydrous ethanol, hydrous ethanol with 10% water content per volume and water. Tests were first carried out at ambient conditions of 20 °C, 1 bar with 150 bar injection pressure using high-speed spray imaging. Then the fuel temperature was lowered to -5 °C, -10 °C and -15 °C to focus on spray formation analysis at realistic cold-start engine conditions. Droplet sizing was also conducted by Phase Doppler Anemometry (PDA). The thermophysical properties of all fuels, including vapour pressure, density, viscosity and surface tension, as well as distillation curves, were obtained over a range of temperatures, and the Reynolds, Weber and Ohnesorge numbers were considered in the analysis. The results revealed the degree of suppression of atomisation for each type of fuel at cold temperatures and effects on spray penetration and cone angle, with ethanol fuels exhibiting the strongest effect in the form of longer initial delay out of the nozzle and shorter penetration.