Flocs surface functional groups evolutions due to an ultrasonic treatment were investigated in respect with the mechanisms involved during sonication. Activated sludge surface functional groups changes were studied after treatment of a sample at different ultrasonic specific energies. Sludge functionality was qualitatively assessed by recording the infrared (FT-IR) spectra of centrifugation pellets. Potentiometric titration coupled with proton surface complexation modeling was used to assess the nature and quantity of ionizable functional groups present at the floc surface and in the aqueous phase. These evolutions were linked to changes of both mixed liquor biochemical composition (TSS, VSS, COD, proteins, humic like substances, polysaccharides) and physical properties (floc size and settleability). Observations carried out showed that activated sludge flocs were essentially mechanically disintegrated by ultrasonic waves: the nature of chemical bonds observed by FT-IR did not shift after ultrasonic treatment. Moreover, the total number of ionizable functional groups measured by potentiometric titrations remained constant during sonication. However, due to the solubilization of organic components induced by cavitation process, the corresponding ionizable functional groups (carboxyl, hydroxyl, amine) were transferred from particulate to soluble fraction. Moreover, due to the variable amount of proteins, humic like substances and polysaccharides solubilised, the relative contributions of carboxyl, hydroxyl and amine groups varied at floc surface.