Exhaust emissions emitted by a non-road mobile machine were studied chasing a tractor in real-world conditions and repeating the same transient tests with a similar engine on an engine dynamometer where additionally, non-road steady state tests were carried out. The engines were equipped with an oxidation catalyst (DOC) and a selective catalytic reduction (SCR) system, and they were fuelled by fossil diesel fuel with ultra-low sulphur content and hydrotreated vegetable oil (HVO). By substituting diesel fuel with HVO the on-road emissions of nitrogen oxides (NOx) reduced 20% and particle number 44%, the emission factors being EFNOx = 1.62 ± 0.04 g/kWh and EFN = (28.2 ± 7.8) × 1013 #/kWh. Similar trend was observed for NOx at laboratory although the emissions were somewhat smaller than on-road. In contrast to real-world, in the laboratory experiment the EFN was only 2% smaller with HVO than with diesel, and these emission factors were almost one order of magnitude smaller than observed on-road. The number size distribution and volatility measurements showed that in real-world experiments small nucleation mode particles were formed during uphill and during downhill in engine braking conditions. These were not observed at laboratory. However, nucleation mode particles were observed in the laboratory experiments at high load steady driving conditions. At steady state tests the emissions strongly depended on engine load and engine speed with both fuels.