The present work involved the pretreatment of cultivated microalgal biomass by ultrasonication to improve its suitability as a substrate in microbial fuel cells (MFCs). Ultrasonication efficiently breaks down the cell walls of microalgae, therefore enhancing the accessibility of intracellular organic material by microbes and thussubsequently producing electrons and protons. In the present research work along with the ultrasonic pretreatment of our samples, non-destructive ultrasonic characterization using anultrasonic interferometer has been done, to study the molecular behavior of the microalgae. Scenedesmus sp. shows better molecular behavior and carbohydrate content than Chlorella sp. Around 47.9 % carbohydrate content estimated in Scenedesmus sp. Hence, Scenedesmus sp. was used as a substrate in the MFC for bioelectricity generation. Electrochemical techniques like electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and polarization studies were done to evaluate the performance of MFCs in the presence of electro-active bacteria Pseudomonas aeruginosa. A maximum power density of 11.64 W/m