Diesel engine emissions are an important source of ultrafine particulate matter (PM) in both ambient air and many occupational settings. Biodiesel is a popular, ‘green’ alternative to petroleum diesel fuel, but little is known about the impact of ‘real world’ biodiesel combustion on workplace PM concentrations and particle characteristics including size, morphology, and composition; or on biological responses. The objectives of the present work were to characterize PM workplace concentrations and tailpipe emissions produced by the combustion of commercially purchased low sulfur petrodiesel and a waste grease B20 blend (20% biodiesel/80% petrodiesel by volume) in heavy duty diesel (HDD) nonroad equipment operating in a ‘real world’ rural recycling center. Furthermore, we assessed the in vitro responses of cell lines representing human lung epithelial cells (BEAS-2B) and macrophages (THP-1) after 24 h of exposure to these real-world particles. Compared to petroleum diesel, use of B20 in HDD equipment resulted in lower mass concentrations of PM2.5, PM<0.25 (particle diameter less than 2.5 and 0.25 micrometer, respectively), and elemental carbon. Transmission electron analysis of PM showed that primary particle size and morphology were similar between fuel types. Metals composition analysis revealed differences between fuels, with higher Fe, Al, V, and Se measured during B20 use, and higher As, Cd, Cu, Mn, Ni and Pb concentrations measured during petrodiesel use. In vitro responses varied between fuels but data supported that waste grease B20 particles elicited inflammatory responses in human macrophages and lung epithelial cells comparable to petrodiesel particles. However, the effects were more pronounced with B20 than petrodiesel at the same mass concentration. Since the primary particle size and morphology were similar between fuels, it is likely that the differential results seen in the in vitro assays points to differences in the composition of the PM. Future research should focus on the organic carbon and metals speciation and potential impact of real world particles on reactive oxygen species generation and mechanisms for differences in the cellular inflammatory responses.