The current paper investigates the spray and combustion characteristics of hydrotreated vegetable oil (HVO), petrol diesel (EN590), blends of HVO with petrol diesel (70% EN590 and 30% HVO), and rapeseed oil methyl esters (RME) in an optically accessible compression ignition engine. Mie scattering and natural luminosity imaging are employed to measure the liquid spray and combustion behaviors. The spray and combustion processes are divided into four stages based on optical imaging. The morphology and quantitative analysis based on imaging provides a method for visualizing the in-cylinder spray and combustion behavior with four test fuels. The ignition delay and combustion characteristics detected from optical measurements are compared to those determined from cylinder pressure. The results show that the ignition delay of HVO and RME occurs earlier and the flame propagation at the premixed combustion stage proceeds faster compared to EN590 and HVO30. The spray and combustion characteristics of HVO30 are similar to EN590. However, ignition occurs earlier for HVO30 due to the higher CN. Comparison of the HVO and RME shows that there is a marginal difference in the ignition delay for these two fuels. However, the combustion duration of RME is shorter than that of HVO.