This paper investigates the bacterial microbiota in tilapia fillets under cold (4 °C), iced (0 °C), and superchilled (-3 °C) storage conditions. At 4 °C, at least seven species/strains of Pseudomonas were detected in the fillets, five of which were dominant either at a certain stage or throughout the entire storage period. Shewanella was less dominant than Pseudomonas at 4 °C, while Serratia became dominant after 6 days storage at 4 °C. The microbiota in fillets stored at 0 and -3 °C were very similar and rarely changed during storage, yet differed greatly from the microbiota at 4 °C. Only two Pseudomonas species/strains grew at 0 and -3 °C, one of which was the most dominant. A Vibrionimonas sp. not found at 4 °C was found to be the second most dominant species at 0 and -3 °C. Shewanella and Psychrobacter were also present at 0 and -3 °C but were the minor genera. The most dominant strains at -3, 0, and 4 °C were separately isolated and subjected to full length 16S rDNA sequencing, which demonstrated that they were identical and were Pseudomonas fluorescens. The changes of the total bacterial count and TVBN value of the fillets inoculated with the isolated P. fluorescens were very similar to those of fillets with natural microbiota. This implies that P. fluorescens is the most important spoiler of tilapia fillets at -3, 0, or 4 °C. PRACTICAL APPLICATION: This research shows that fewer species of bacteria survive at 0 and -3 °C than those at 4 °C, while among these bacteria, the most important spoiler is P. fluorescens. This may provide some clues to extend the shelf life of tilapia fillets by taking some inhibitory measures targeted at P. fluorescens in the future.