Abstract: Objective: To explore the antibacterial activity of Solenocera crassicornis processing by-products fermentation (SPBF) on specific spoilage organisms in the late stage of squid refrigeration (SR-SSOs). Method: Solenocera crassicornis by-products including shrimp heads and shells were fermented with Lactobacillus fermentum to prepare SPBF. The molecular weight distribution of peptidic fractions in SPBF was analyzed. The antibacterial effect of SPBF against SR-SSOs, and membrane leakage and damage of SR-SSOs treated by SPBF were determined. Furthermore, 16S rRNA sequencing technology was used to reveal the impact of SPBF on the microbiota diversity of SR-SSOs, as well as the pathways of antibacterial action of SPBF were predicted. Results: The relative percentages of peptidic fractions >5000 Da, 5000~1000 Da, 1000~500 Da, and <500 Da in SPBF were 34.61%, 1.57%, 5.63%, and 58.19%, respectively. The diameter of the inhibition zone of SPBF against SR-SSOs reached 14.3±0.8 mm, and the minimum inhibitory concentration was 1.89 mg/mL (measured by peptide concentration). SR-SSOs did not show significant leakage of intracellular genetic material after 12 to 48 hours of SPBF treatment. However, the peptide concentration of SPBF decreased, and further reduced with action time increased. After 12 hours of SPBF treatment, a large number of bacterial surfaces of SR-SSOs became irregular and concave. However, the membrane structure of SR-SSOs did not undergo complete lysis. The relatively dominant strains in SR-SSOs were Brochothrix thermosphacta (84.68%) and Carnobacterium maltaromaticum LMA28 (5.20%). After 12 hours of SPBF treatment, the dominant species in SR-SSOs were Carnobacterium maltaromaticum LMA28 (16.63%), Carnobacterium divergens (5.00%), and Pseudomonas lundensis (3.55%). LEfSe linear discriminant analysis (LDA) revealed that the strains of Brochothrix thermosphacta, Psychrobacter, Pseudomonas lundensis, Carnobacterium maltaromaticum LMA28, and Carnobacterium divergens could significantly distinguish the microbiota composition between SPBF treatment group and SR-SSOs. Compared with SR-SSOs, SPBF significantly reduced the expression levels of carbohydrate metabolism, transcription, nucleotide metabolism, as well as replication and repair pathways in the bacteria after 12 hours of action, while significantly increasing the levels of pathways involved in membrane transport and energy metabolism (P<0.05). Conclusions: SPBF can inhibit SR-SSOs, especially Brochothrix thermosphacta, through a non-membrane damage mechanism, which may be speculated to be related to the effective antibacterial peptides in SPBF being transported into SR-SSOs through membrane, thereby inhibiting carbohydrate metabolism and genetic material production.