It is well known that direct injection (DI) is a technology enabler for stratified combustion in spark-ignition (SI) engines. At full load or wide-open throttle (WOT), partial charge stratification can suppress knock, enabling greater spark advance and increased torque. Such split-injection or double-pulse injection strategies are employed when using gasoline in DI (GDI). However, as the use of biofuels is set to increase, is this mode still beneficial? In the current study, the authors attempt to answer this question using two gasoline-alternative biofuels: firstly, ethanol; the widely used gasoline-alternative biofuel and secondly, 2,5-dimethylfuran (DMF); the new biofuel candidate. These results have been benchmarked against gasoline in a single-cylinder, spray-guided DISI research engine at WOT (λ = 1 and 1500 rpm). Firstly, single-pulse start of injection (SOI) timing sweeps were conducted with each fuel to find the highest volumetric efficiency and IMEP. The resulting optimum SOI timing for gasoline was then used as the first injection (SOI₁) with each fuel in the split-injection tests. In this instance, second SOI timing (SOI₂) sweeps were made using two split-ratios (SOI₁:SOI₂ = 1:1 and 2:1). For the single-pulse SOI timing sweeps, the change in IMEP when using ethanol is symmetrical either side of the maximum. However, when using gasoline and DMF, the behavior is asymmetrical, with maximums later and earlier than with ethanol, respectively. For split-injection, the maximum IMEP increases when fuelled with the biofuels, whilst maintaining acceptable engine stability. This increase, however, is much more dependent on SOI₂ timing than with gasoline, due to the deterioration of in-cylinder mixing and slower combustion.