Pseudomonas fragi and Serratia liquefaciens are key spoilage microorganisms in aerobically packed groupers during cold storage. Spoilage bacteria were inoculated into fillets to investigate their spoilage potential. The results showed that P. fragi had stronger degradation activity, and the P. fragi inoculated group had higher total volatile basic nitrogen (TVB-N) and K values, lower water holding capacity (WHC), and textural qualities. The P. fragi inoculated group had higher levels of carbonyl groups and lower levels of sulfhydryl groups, indicating higher levels of protein oxidation. The secondary and tertiary structure of myofibrillar protein (MP), scanning electron microscopy, and low-field nuclear magnetic resonance (LF-NMR) analyses showed that P. fragi degraded myofibrils more and induced water loss from the muscle. Metabolomics analyses indicated that purine metabolism and glycine, serine, and threonine metabolism were important pathways associated with P. fragi spoilage; linoleic acid metabolism and β-alanine metabolism were important pathways for S. liquefaciens spoilage.