With the slow but ineluctable depletion of fossil fuels, several avenues are currently being explored in order to define the strategic boundaries for a clean and sustainable energetic future, while accounting for the specificities of each sectors involved. In regard to transport applications, alternative fuels may represent a promising solution, at least at short or middle term, such as the International Energy Agency foresees that their share could account for 9% of the road transport fuel needs by 2030 and 27% by 2050, with the potential resources to reach 48% beyond. If they have already been included in significant blending proportions with conventional fossil fuel in most of the occidental countries, their introduction also coincides with a longtime established program of continuously more drastic standards for engine emissions of NOx and PM, now even further demanding by the seek for combustion efficiency aiming at reducing CO2 emissions. While several works discuss the alternative fuels effect on exhaust emissions when used directly in production Diesel engines, results and analysis are sometimes contradictory, depending sometimes on the conditions in which they were obtained, and the causes of these results remain unclear. Therefore, in order to better understand their effect on the combustion processes, and thus extract the maximum benefits from these fuels in the optimization of engine design and calibration, a detailed comprehension of their spray and combustion characteristics is essential. The approach of this study is mostly experimental and based on an incremental methodology of tests aiming at isolating injection and combustion processes with the objective to identify and quantify the role of both fuel physical and chemical properties at some key stages of the Diesel combustion process. After obtaining a detailed characterization of their properties, five fuels have been injected in an optical engine enabling a sharp control of the thermodynamic environment, and the application of optical techniques for spray measurements and characterization. Diagnostics applied to free jets in inert conditions allowed to discuss the fuel effect on the processes of atomization, vaporization and development of the mixture fraction field, while the reactive environment, by including the previous findings, enabled to assess their effect on ignition, soot formation and flame temperature. As a result, empirical models have been developed in order to predict the liquid length of these fuels based on their properties measured by standards at ambient conditions. These correlations confirmed the minor effect of liquid atomization for fuel vaporization as stated in the Siebers mixing-limited hypothesis. They also revealed the significance of fuel latent heat well correlated by the fuel density. By extension of results obtained in another laboratory, the equivalence ratio at any point of the inert spray showed to be likely modified by the unique difference in stoichiometry among fuels. Under reactive conditions, the key role played by fuel ignitability on the lift-off length establishment has been confirmed as well as the role of the latter on soot formation. The increase of flame sooting propensity showed to cool down the flame and produce lower flame temperature, a priori in favor of decreasing thermal NOx formation. Such chain-reaction between ignition, lift-off, soot formation and flame temperature was already suggested in the literature although in separate studies. For the first time, these relationships were associated in the same test campaign with the objective to assess the effect of fuel properties on the spray reaction of combustion. By permitting a better understanding of the fuel effect and its consequences on key stages of the Diesel combustion, this study provides arguments to discuss the emissions results obtained with real production engines and beyond, also provides a database of both inputs and outputs for comparison with spray and combustion modeling. In that sense, it participates to the global effort on the way to improve the design of future engines in terms of efficiency and contamination.