Fuels from lignocellulosic biomass have the potential to contribute to sustainable future mobility targets by reducing the fossil CO2 emissions of the transport sector. Of special interest for the diesel engine are oxygenated fuels, since they can help to solve traditional conflicts of objectives like the soot–NOx trade-off or the efficiency–NOx compromise. Dibutyl ether (DBE) and oxymethylene ethers (OME) are among the most promising fuel candidates. The suitability of these compounds for diesel engines is investigated in this study. The fuels are injected in pure form as well as a diesel–biofuel blend with 20% volumetric biogenic share. During the course of these investigations special attention is given to soot and particle emissions, and also to measured engine efficiency. The combustion tests are combined with an analysis of suitable production paths of the evaluated bio-ethers as second generation biofuels. Production simulation shows high greenhouse gas savings potential, but also high investment costs.