Effects of ultrasonic pretreatment and packing carriers on sludge reduction, settleability, dewaterability and microbial community structure in the anaerobic side-stream reactor (ASSR) were investigated with three anaerobic reactors operated in parallel. Ultrasonication from 3.65% in the ASSR to 5.08%, and packing carriers further enhanced the efficiency to 19.2%. Ultrasonic pretreatment of sludge decreased oxidation-reduction potential in ASSR and enhanced the release of intracellular substances. The deterioration of sludge settleability and dewaterability in the ASSR after ultrasonic pretreatment was improved by packing carriers. Illumina-MiSeq sequencing showed that microbial richness and diversity increased after ultrasonic pretreatment and packing carriers in the ASSR. Packing carriers favored the growth of slow grower (Dechloromonas), fermentative bacteria (Draconibacteriaceae, Fusibacter, Acidaminobacter) and floc-forming bacteria (Zoogloea), while hydrolytic and predatory bacteria (Saprospiraceae_unculture) and slow grower (Thauera) was enriched in the ASSR.