Third-generation feedstocks and its constituent biofuels have shown promising results in the light of sustainable production and as a feasible fuel source for internal combustion (IC) engines. Hence, in this study, a third-generation microalgae feedstock (Scenedesmus quadricauda) biomass was cultivated sustainably using an in situ tubular photo bioreactor and raceway pond to synthesize quintet carbon chained amyl alcohol using Ehrlich biosynthetic pathway. On analyzing the synthesized amyl alcohol, a homogenous mixture of a 20% (vol/vol) amyl alcohol-diesel blend showed similarities with conventional diesel in their physio-chemical properties. This potential fuel source was analyzed though systematic experimentation at maximum throttle position condition in a light commercial vehicle compression ignition engine. The conducted experiments were directed by response surface methodology (RSM) coupled with central composite design (CCD) which delivered a set of influential and interactive responses on engine testing. At optimal operating condition, 0.7% rise in brake thermal efficiency (BTE) and an increased specific fuel consumption of 5.6% is reported due to the lower heating value of the biofuel. Furthermore, a 55.8% and 5.4% drop in smoke and carbon monoxide emissions is observed. However, oxides of nitrogen emission increases by 31.7% for biofuel operation as a tradeoff for the improved combustion characteristics achieved.