Bio-butanol has been considered as a promising alternative fuel for transportation due to its advantageous physical and chemical properties, but the high recovery and dehydration cost reduced its competitiveness in the fuel market. In order to achieve the bio-fuel advantage, the current work tried to apply and evaluate the upstream products of bio-butanol, Isopropanol-Butanol-Ethanol (IBE). These mixtures are obtained by ABE fermentation and IBE fermentation respectively, as alternative diesel fuel. Isopropanol-Butanol-Ethanol (IBE) is an intermediate product in the IBE fermentation process for producing bio-butanol. The study was carried out in an AVL 5402 single-cylinder diesel engine fueled by IBE-Diesel, ABE-Diesel and neat diesel. During the experiments, the engine speed, engine loads, and injection timings were varied. The results showed that with the addition of IBE in diesel fuel, the engine can still operate smoothly with a set pilot injection. The combustion phasing of IBE20 was retarded and the premixed combustion was enhanced in comparison with that of neat diesel, but the difference of two bio-fuel upstream products did not show much difference. Pilot and main injection timing influenced the heat release process of IBE-Diesel blends more significantly than that of neat diesel. Thus, injection strategy might be a potential way to control the combustion of bio-fuel upstream products as alternative diesel fuel. While the IBE NOx emissions were typically lower than that of diesel, diesel and IBE shared similar PM emissions.