Biofuels have been considered to be sustainable energy source and one of the major alternatives to petroleum-based road transport fuels due to a reduction of greenhouse gases emissions. However, their effects on urban air pollution are not straightforward. The main objective of this work is to estimate the emissions and energy use from bio-fuelled vehicles by using an integrated and flexible modelling approach at the urban scale in order to contribute to the understanding of introducing biofuels as an alternative transport fuel. For this purpose, the new Traffic Emission and Energy Consumption Model (QTraffic) was applied for complex urban road network when considering two biofuels demand scenarios with different blends of bioethanol and biodiesel in comparison to the reference situation over the city of Coimbra (Portugal). The results of this study indicate that the increase of biofuels blends would have a beneficial effect on particulate matter (PM2.5) emissions reduction for the entire road network (−3.1% [−3.8% to −2.1%] by kg). In contrast, an overall negative effect on nitrogen oxides (NOx) emissions at urban scale is expected, mainly due to the increase in bioethanol uptake. Moreover, the results indicate that, while there is no noticeable variation observed in energy use, fuel consumption is increased by over 2.4% due to the introduction of the selected biofuels blends.