The (partial or total) substitution of petro-diesel with biodiesel in internal combustion engines (ICEs) could represent a crucial path towards the decarbonization of the energy sector. However, critical aspects are related to the controversial issue of the possible increase in Nitrogen Oxides (NOx) emissions. In such a framework, the proposed study aims at investigating the effects of biodiesel share and injection timing on the performance and NOx emissions of a diesel micro combined heat and power (CHP) system. An experimental campaign has been conducted considering the following operating conditions: (i) a reference standard injection timing (17.2° BTDC), an early injection timing (20.8° BTDC), and a late injection timing (12.2° BTDC); (ii) low (0.90 kW), partial (2.45 kW), and full (3.90 kW) output power load; and (iii) four fuel blends with different biodiesel (B) shares (B0, B15, B30, and B100). Experimental data were also elaborated on thanks to the response surface modelling (RSM) technique, aiming at (i) quantifying the influences of the above-listed variables and their trends on the responses, and (ii) obtaining a set of predictive numerical models that represent the basis for model-based design and optimization procedures. The results show: (i) an overall improvement of the engine performance due to the biodiesel presence in the fuel blend —in particular, B30 and B100 blends have shown peak values in both electrical (29%) and thermal efficiency (42%); (ii) the effective benefits of late SOI strategies on NOx emissions, quantified in an overall average NOx reduction of 27% for the early-to-late injection, and of 16% for the standard-to-late injection strategy. Moreover, it has emerged that the NOx-reduction capabilities of the late injection strategy decrease with higher biodiesel substitution rates; through the discussion of high-prediction-capable, parametric, data-driven models, an extensive RSM analysis has shown how the biodiesel share promotes an increase of NOx whenever it overcomes a calculated threshold that is proportional to the engine load (from about 66.5% to 85.7% of the biodiesel share).