The growing concern about air quality and the impact exhaust particles can have on the environment has resulted in the increased use of alternative fuels. A sampling campaign from a conventional heavy diesel engine operated in typical transient cycle or steady-state condition, and running on diesel, 30% biodiesel in diesel, and 100% biodiesel was carried out. The particulate composition was characterized using Fourier Transform Infrared (FTIR) spectroscopy, Two-step Laser Mass Spectrometry (L2MS), Secondary Ion Mass Spectrometry (SIMS), thermo-optical analysis, and capillary electrophoresis. Elemental carbon is demonstrated to decrease from diesel to 100% biodiesel, in agreement with the evolution of aromatic bands and the MS abundance of Cn– fragments, while organic carbon exhibits a constant level irrespective of the working regime. Aliphatic, aromatic, carboxyl, carbonyl, hydroxyl functionalities, and nitro compounds are found to depend on the engine-working regime. Mass spectra are mainly characterized by alkyl fragments (CnH2n+1+), associated to normal and branched alkanes, PAHs and their alkylated derivatives. The addition of biodiesel to diesel changes the particulate composition towards more oxygenated constituents, such as carbonyl groups attributed to methyl ester CH3O+ fragments of unburned biodiesel. Fuel-specific fragments have been identified, such as C3H7O+ for diesel, and C2H3O2+ and CH3O– for biodiesel. Nitrogenized compounds are revealed by -NO2 functionalities and N-containing fragments. Principal Component Analysis (PCA) was successfully applied to discriminate the engine operating conditions, with a higher variance given by the fuel, thus allowing to better evaluate the environmental impacts of alternative energy source emissions.