This work suggests an interpretation to the quantitatively higher formation of NOx in a compression ignition (CI) engine when fueled with pure biodiesel (B100). A comparative study about the use of rapeseed oil methyl ester (RME) and diesel fuel mixtures on injection timing, in-chamber pressure, heat release rate, and NOx emissions were carried out using a diesel engine equipped with a pump-line-nozzle injection system. Such engines are still widely adopted mainly in agriculture, as the fleet of agricultural machinery is particularly old (often over 20 years) and the use of biofuels can reduce the environmental footprint of the sector. This work aims to supply some general explanations and figures useful to interpret the phenomena occurring within the fuel line and in the combustion process when using biodiesel, as well as in engines with different construction characteristics and fueling systems. Given the contradictory results available in the literature, the so-called “biodiesel NOx effect” cannot be explained solely by the different physical properties of biodiesel (in particular, a higher bulk modulus). Experimental results show that, with the same pump settings, the start of injection with the RME is slightly advanced while the injection pressure values remain almost the same. With the RME, the pressure in the injection line increases faster due to its greater bulk modulus but the pressure rise starts from a lower residual pressure. The start of combustion takes place earlier, the heat release during the premixed phase is steeper, and a higher peak is reached. The NOx emissions with the RME are at least 9% higher when compared to mineral diesel fuel. The greater amount of the RME injected per cycle compensates for its minor lower heating value, and the brake torque at full load is similar to the two analyzed fuels. Finally, a variation of the pump line timing is evaluated in order to assess the effect of the delay and the advance of the injection on the performance of the engine and on the emissions. A viable and simple solution in the variation of the injection strategy is suggested to counterbalance the biodiesel NOx effect.