The utilisation of internal combustion engines is one of the main causes of particle emissions in urban areas. As the interest for the utilisation of biofuels increases, it is important to understand their effect on particle number emissions. In this paper, the particle size distribution and the particle number emissions from a gasoline-optimised direct-injected spark-ignited (DISI) engine are investigated. The effects of five different biofuel alternatives on these emissions were evaluated and compared to gasoline. The utilisation of the high-resolution, high-temperature ELPI+ enabled undiluted measurements of the particle size distribution down to 6 nm, without extensive cooling of the engine exhaust. Contrary to other studies, the results show that the particle number emissions for the three measured cut-off sizes (23, 10 and 7 nm) increased with the utilisation of oxygenated biofuels. The results indicate that the decreased volatility and energy density of the alcohols has a more significant impact on the particle formation in a DISI engine than the increased oxygen content of these fuels.