Single Fuel Policy arose as a necessity in order to fulfill an effectiveness of the used equipment through the use of a single fuel in the battlefield. Thus, it was first ensured that the fuel's specification was standard with the equivalent commercial fuel which is still in use in the NATO countries and of course that the fuels' chemical characteristics allow it to be imported, stored, carried and distributed through the NATO oil piping system. So, in 1998 the NATO countries agreed to adopt the use of F-34 as a single fuel. Furthermore, the atmospheric pollution, which is one of the greatest problems that concerns especially the vehicle industries, drove the research on fuels to bio-fuels, like bio-diesel. Bio-diesel is an alternative fuel, which can be used by a diesel engine, even a military one, without a beforehand modification. It is produced by natural, renewable sources, like vegetable oil and animal fat. International bibliography includes a great amount of information concerning the effect of the use of bio-diesel on performance and pollutant emissions (Oxides of Nitrogen, Carbon Monoxide, unburnt Hydrocarbons and Soot) of a Direct Injection (DI) diesel engine. In this respect, an experimental investigation has been conducted in the laboratory of Thermodynamic and Propulsion Systems at the Hellenic Air Force Academy. The main scope of the present investigation is to evaluate the use of F-34 and bio-diesel as a full substitute for diesel fuel in a direct injection diesel engine. Thus, the present work covers a range of engine load and speed operating conditions under bio-diesel, F-34 and normal diesel operation (baseline conditions), comprising measurements of engine power output, fuel consumption, exhaust gas temperature and the concentrations of exhaust gas emissions (Nitrogen Oxide and Soot). Through this work it is shown that for all test cases examined when diesel fuel is full substituted by F-34 no significant effect is observed on the basic operating parameters and the brake specific fuel consumption as well while it seems to affect the maximum combustion pressure and the ignition delay period. As far as the exhaust emissions are concerned, the full substitution of diesel fuel with F-34 has a negligible influence on the concentrations of Nitrogen Oxide emissions while it seems to affect seriously the values soot emission concentrations. As far as the behavior of bio-diesel compared with diesel's is concerned, the results revealed a slight increase in the exhaust gas temperature, an increase in the brake specific fuel consumption, which at low loads tends to be identified with the diesel's, a decrease in soot emissions and brake thermal efficiency, and finally a slight decrease in NO emissions at low loads, while at full load NO emissions are considerable higher.