Most microbial time–temperature indicators (TTIs) considered only one spoilage strain. This research compared single and dual spoilage strains‐based microbial TTI for quality changes of chilled grouper fish (Epinephelus fuscoguttatus x E. lanceolatus) fillet products during distribution. The next‐generation sequencing (NGS) and traditional plate count approach showed that Pseudomonas fragi and Vibrio parahaemolyticus were specific spoilage bacteria at 7 and 15°C. A dual‐strain TTI response provides more accurate results than a single‐strain TTI and provides an irreversible color change from yellow to reddish‐brown, showing levels of fish freshness. The microbial TTI comprises fish spoilage bacteria strains with 3 log CFU/ml, a nutrient broth supplemented with 2% NaCl as a medium, and phenol red with 0.25 mg/ml as a pH indicator. Overall, this study points to the applicability of a dual‐strain microbial TTI as a valuable tool for monitoring fish quality changes during cold chain break condition.