A single-cylinder marine diesel engine was modified to be operated in reactivity controlled compression ignition (RCCI) combustion mode. The engine fueling system was upgraded to a common rail fuel injection system. Natural gas (NG) was used as port fuel injection, and a diesel/sunflower methyl ester biodiesel mixture was used for direct fuel injection. The fraction of biodiesel in the direct fuel injection was changed from 0% (B0; 0% biodiesel and 100% diesel) to 5% (B5) and 20% (B20) while keeping the total energy input into the engine constant. The objective was to understand the impacts of the increased biodiesel fraction on the combustion characteristics and stability, emissions, and knocking/misfiring behavior, keeping all other influential parameters constant. The results showed that nitrogen oxides (NOx) emissions of B5 and B20 without the need for any after-treatment devices were lower than the NOx emission limit of the Euro VI stationary engine regulation. B5 and B20 NOx emissions decreased by more than 70% compared to the baseline. Significantly more unburned hydrocarbons (UHCs) and carbon monoxide (CO) emissions were produced when biodiesel was used in the direct fuel injection (DFI). The results also showed that using B5 and B20 instead of B0 led to an increase of 18% and 13.5% in UHCs and an increase of 88.5% and 97% in CO emissions, respectively. Increasing the biodiesel fraction to B5 and B20 reduced the maximum in-cylinder pressure by 3% and 10.2%, respectively, compared to B0. Combustion instability is characterized by the coefficient of variation (COV) of the indicated mean effective pressure (IMEP), which was measured as 4.2% for B5 and 4.8% for B20 compared to 1.8% for B0. Therefore, using B20 and B5 resulted in up to 34.9% combustion instabilities, and 18.5% compared to the baseline case. The tendency for knocking decreased from 13.7% for B0 to 4.3% for B20. The baseline case (B0) had no misfiring cycle. The B5 case had some misfiring cycles, but no knocking cycle was observed. Moreover, the historical cyclic analysis showed more data dispersions when the biodiesel fraction increased in DFI. This study shows the potential of biodiesel replacement in NG/diesel RCCI combustion engines. This study shows that biodiesel can be used to effectively reduce NOx emissions and the knocking intensity of RCCI combustion. However, combustion instability needs to be monitored.