In order to further develop and utilize shrimp processing by-products, in this study, a novel antibacterial hydrolysate of shrimp by-products by pepsin hydrolysis (SPH) was prepared. The antibacterial effect of SPH on specific spoilage organisms of squid after end storage at room temperature (SE–SSOs) was investigated. SPH showed an antibacterial effect on the growth of SE–SSOs, with (23.4 ± 0.2) mm of inhibition zone diameter. The cell permeability of SE–SSOs was enhanced after SPH treatment for 12 h. Some bacteria were twisted and shrunk, while pits and pores formed and intracellular contents leaked under scanning electron microscopy observation. The flora diversity of SE–SSOs treated with SPH was determined by a 16S rDNA sequencing technique. Results showed that SE–SSOs were mainly composed of the phyla of Firmicutes and Proteobacteria, among whichParaclostridium(47.29%) andEnterobacter(38.35%) were dominant genera. SPH treatment resulted in a significant reduction in the relative abundance of the genusParaclostridiumand increased the abundance ofEnterococcus. Linear discriminant analysis (LDA) of LEfSe conveyed that SPH treatment had a significant impact on altering the bacterial structure of SE–SSOs. The 16S PICRUSt of Cluster of Orthologous Group (COG) annotation revealed that SPH treatment for 12 h could significantly increase the function of transcription level [K], while SPH treatment for 24 h could downregulate post-translational modifications, protein turnover, and chaperone metabolism functions [O]. In conclusion, SPH has a proper antibacterial effect on SE–SSOs and can change the flora structure of SE–SSOs. These findings will provide a technical basis for the development of inhibitors of squid SSOs.