The effects of different inoculation methods of two dominant spoilage bacteria on the quality change in obscure pufferfish (Takifugu obscurus) during cold storage and their spoilage ability were evaluated. Sterile whole fish were inoculated with Pseudomonas fluorescens, Shewanella putrefaciens, or their combination. Samples treated with sterile water were taken as the control group. All samples were stored at 4 ℃. Microbial (total viable count, Pseudomonas bacteria count, and Shewanella bacteria count), physiochemical (pH, total volatile base nitrogen (TVB-N)) contents, K value, and xanthineoxidase (XOD) activity), and protein properties (total sulfhydryl (SH) content, myofibril fragmentation index (MFI), and intrinsic fluorescence intensity (IFI)) were measured at 2-day intervals during storage, and magnetic resonance imaging (MRI) was also carried out to analyze quality changes comprehensively. The spoilage ability of the dominant spoilage bacteria was evaluated by the yield factor of spoilage metabolite production. The results showed that total viable count, TVB-N contents and K value in the inoculation groups were higher than those of the control group. Inoculation with the spoilage bacteria accelerated protein oxidation and promote the breakage of myofibril, resulting in changes in the tertiary structure of fish proteins and a significant decrease in water content. Moreover, the rate of quality deterioration in the combined inoculation group was faster than that in the single inoculation groups. According to the results of the yield factor of spoilage metabolite production, the combined inoculum had the strongest spoilage ability, followed by S. putrefaciens, and P. fluorescens had the weakest spoilage ability.