Fish is an important source of antimicrobial peptides. This study aimed to identify and screen antibacterial peptides with excellent antibacterial activity derived from sturgeon spermary peptides (SSPs) and to analyze their antibacterial activity and mechanism. Liquid chromatography-mass spectrometry/mass spectrometry methods were used to analyze and identify peptide sequences, computational prediction tool and molecular docking methods were used for virtual screening of antimicrobial peptides, and finally, candidate peptides were synthesized by solid-phase synthesis method. The results demonstrate that SSPs have excellent inhibitory activity against with an inhibitory rate of 76.46%. Most parts of the SSPs were derived from the sturgeon () histones, and the coverage of histone H2B was the highest (45%). Two novel peptides (NDEELNKLM and RSSKRRQ) were obtained by in silico prediction tools and molecular docking, which may interact with the DNA gyrase and dihydrofolate reductase of by forming salt bridges and hydrogen bonds. Compared to the individual peptides, the antibacterial effect was significantly improved by mixing the two peptides in equal proportions. Two novel peptides change the permeability of the cell membranes and may exert antimicrobial activity by inhibiting the metabolic process of the nucleic acids.