Increasing the biodiesel content of diesel fuels is encouraged because of its reduced carbon footprint. Pure rapeseed methyl ester (RME)and used cooking oil methyl ester (UCOME) are characterised by well-to-tank greenhouse gas (GHG) reductions of 54% and 88% compared to pure B0 petrodiesel, respectively. Captive fleets such as public transport buses could benefit from these GHG reductions by increasing the biodiesel content of their fuel because they have a consequent yearly fuel consumption. The aim of this paper is to compare on-road tailpipe emissions of a diesel bus when increasing the biodiesel concentration in the fuel. The tests were carried out on a standard city bus belonging to the Euro V EEV emission standard that was equipped with a portable emission measurement system measuring NO, NO2, PN, CO and CO2 at the tailpipe. The bus followed the SORT which is representative of urban bus driving. The heavy urban on-road measurements indicated increased NOx emissions (24–26%), decreased PN emissions (43–45%) and slightly decreasing CO emissions for B30 RME and UCOME compared to B7. A measurement uncertainty analysis showed that the CO emissions were less reliable. Similar conclusions were drawn for the easy urban on-road bus emission measurements with smaller differences between B7 and B30 RME and UCOME.