Due to its strong impact on health, particulate matter is increasingly regulated by government emission standards for vehicles. As one of the sources of particulate matter is the soot produced by internal combustion engines, it remains a challenge to improve advanced combustion modes to reduce it. There is still, however, some lack of understanding about the formation and oxidation processes of soot, especially in “realistic” conditions, such as for example at high temperature and pressure conditions with or without the presence of exhaust gases. The objective of this study is to investigate soot formation in the case of n-Dodecane spray flames at conventional Diesel engine conditions generated in the New One Shot Engine by using diffused back-illumination extinction with different CO2 and water vapour contents. It was found that CO2 addition reduces the soot mass fraction if its volumetric concentration in ambient mixtures is at least 4.5% while 1% of water is sufficient to significantly reduce the soot mass fraction. The impact of the ambient mixture obtained in ECN spray A pre-burn vessels was also investigated to assess the accuracy against soot measurements available in the literature.