Bio-butanol has been considered as a promising alternative fuel for internal combustion engines due to its advantageous physicochemical properties. However, the further development of bio-butanol is inhibited by its high recovery cost and low production efficiency. Hence, the goal of this study is to evaluate two upstream products from different fermentation processes of bio-butanol, namely acetone-butanol-ethanol (ABE) and isopropanol-butanol-ethanol (IBE), as alternative fuels for diesel. The experimental comparison is conducted on a single-cylinder and common-rail diesel engine under various main injection timings (MIT) and equivalent engine load (EEL) conditions. The experimental results show that ABE and IBE significantly affect the combustion phasing. The start of combustion (SOC) is retarded when ABE and IBE are mixed with diesel. Furthermore, the ABE/IBE-diesel blends are more sensitive to the changes in MIT compared with that of pure diesel. When the MIT is advanced, the peak in-cylinder pressure and heat release rate (HRR) of ABE20 and IBE20 are lower than that of pure diesel but eventually are higher. Brake thermal efficiency is slightly reduced with the addition of ABE or IBE into diesel under tested conditions. But the reduction for IBE20 is not noticeable. Both ABE and IBE mixtures show a potential to reduce NOx and soot emissions. However, the NOx emissions from the ABE and IBE mixtures are slightly higher than that of pure diesel when the MIT is advanced to 8°CA BTDC. These results indicate that ABE/IBE-diesel blends, coupled with an optimized injection, can effectively control the exhaust emissions of diesel engines.