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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.