The increasing share of using biofuels in vehicles (mandated by current regulations) leads to a reduction in particle size, resulting in increased particle toxicity. However, existing regulations disregarded small particles (sub-23 nm) that are more toxic. This impact is more significant during vehicle cold-start operation, which is an inevitable frequent daily driving norm where after-treatment systems prove ineffective. This study investigates the impact of biofuel and lubricating oil (as a source of nanoparticles) on the concentration, size distribution, median diameter of PN and PM, and their proportion at size ranges within accumulation and nucleation modes during four phases of cold-start and warm-up engine operation (diesel-trucks/busses application). The fuels used were 10% and 15% biofuel and with the addition of 5% lubricating oil to the fuel. Results show that as the engine warms up, PN for all the fuels increases and the size of particles decreases. PN concentration with a fully warmed-up engine was up to 132% higher than the cold-start. Sub-23 nm particles accounted for a significant proportion of PN (9%) but a smaller proportion of PM (0.1%). The fuel blend with 5% lubricating oil showed a significant increase in PN concentration and a decrease in particle size during cold-start.