Abstract A novel approach to the intensification of renewable and sustainable production of photoautotrophic ethanol from microalgae was investigated using periodic ultrasonication. The effect of utilizing ultrasonic pulsing during ethanol production using a metabolically engineered ethanol producing cyanobacterium Synechocystis PCC6803 strain NAV001 was analysed with an ultrasonic frequency of 20 kHz. Ultrasonic treatment resulted in enhancement of ethanol yields. The ethanol yield was found to be higher when ultrasonic pulsing was initiated during the exponential phase of growth. The optimum ultrasonic conditions for enhanced yield of ethanol within this model system was found to be 30 °C, 15% of power input (97.5 W) and 10 min pulse time. The cytotoxic effect of ultrasonic pulsation was investigated by analysing the survival percentage and auto-fluorescence of the cyanobacterium via imaging by confocal laser scanning microscopy. The overall effect of ultrasonic dose on ethanol production and growth rate of microalgae was analysed by using Haldane inhibition kinetics.