Changes in the microbial diversity of golden pompano (Trachinotus ovatus) under different temperature conditions, including room temperature (RT, 20 °C), refrigerated temperature (RF, 4 °C), and supercooling preservation temperature (SP, −1 °C), were investigated using high-throughput sequencing (HTS). The results showed that the total viable count (TVC) and total volatile basic nitrogen increased as the storage period was extended. TVC results suggested that the shelf-life of golden pompano was 30 h, 8 days, and 21 days for RT, RF, and SP, respectively. HTS analysis of the samples revealed that the microbial community diversity decreased as the storage time increased, and the decrease was delayed by low temperatures. Significant differences at the genus level of the dominant bacteria were also observed among the groups exposed to different storage conditions. The dominant spoilage bacteria for golden pompano stored at 20 °C was Photobacterium, those of the 4 °C sample were Shewanella, Pseudomonas, and Psychrobacter, and that of the −1 °C sample was Pseudomonas. This study provides a basis for developing strategies to control bacterial growth.