The effects of ozone water (OW) and ultrasound cleaning (UL) on microbial community diversity of crayfish were studied through microbial viable count and 16S rRNA gene sequencing. The results showed that compared with the control (CK), the ozone water combined with ultrasound cleaning (OCU) showed a significant reduction (p < 0.05) in total viable count (TVC), psychrophilic viable count (PVC), mesophilic viable count (MVC), Pseudomonas, hydrogen sulfide-producing bacteria (HSPB), molds and yeasts. Concretely, the TVC of the CK, OW, UL and OCU were 5.09, 4.55, 4.32 and 4.06 log CFU/g, respectively. The dominant bacterium in untreated crayfish was Chryseobacterium, and its relative abundance was reduced by combined treatment. Color measurement and sensory evaluation suggested that a satisfactory sensory experience could be obtained on the crayfish applied with OCU. In brief, OCU could be used as a cleaning strategy to control the microbial quality of crayfish and have no influence on its quality.