Ultrasonication-assisted sludge digestion technology is a lately used alternative sludge treatment method in wastewater treatment plants (WWTPs). This study focused on determining the influence of ultrasonication on aerobic and anaerobic sludge digestion, two most commonly used sludge handling processes, as well as on the investigation of microbial community structure after digestion. The effect of ultrasonication as a pre-treatment technique prior to sludge digestion on microbial population dynamics was not yet investigated comprehensively. Sludge sample taken from the primary and secondary settling tanks of a domestic wastewater treatment plant was used during the experiments. Based on the relevant data, while applied ultrasonication did not improve the anaerobic digestion efficiency, progress was achieved in the sludge dewaterability characteristics at the end of aerobic digestion. According to the results of both denaturant gradient gel electrophoresis and pyrosequencing data, ultrasonic pre-treatment decreased the richness of the microbial population in aerobic digestion, whereas increased the biocomplexity of the population in anaerobic digestion. We revealed that sludge pre-treatment with ultrasonication does not always improve the digestion performance. Composition of the sludge was the main factor defining the digestion performance.