This paper investigates the anti-knock properties of biofuels that can be produced from microorganism metabolic processes. The biofuels are rated using Research Octane Number (RON) and Blending Research Octane Number (BRON), which determine their potential as additives for fuel in spark ignition (SI) engines. Tests were conducted using a single-cylinder Cooperative Fuel Research (CFR) engine and performance of the biofuels was compared to primary reference fuels (PRFs). The investigated fuels include 3-methyl-2-buten-1-ol, 3-methyl-3-buten-1-ol, 2-methylpropan-1-ol (isobutanol), and limonene. Results show that 3-methyl-2-buten-1-ol, 3-methyl-3-buten-1-ol, and 2-methylpropan-1-ol (isobutanol) sufficiently improve the anti-knock properties of gasoline.