Crab roe sauce (CRS) is prone to spoilage due to microbial contamination. Therefore, this study aimed to investigate the inhibitory effects and mechanisms of a mixed bio-preservative (0.025% ε-polylysine hydrochloride (ε-PL) + 0.01% nisin (NS) + 0.01% tea polyphenols (TPs)) on the specific spoilage bacteria (SSB) in CRS. First, the SSB in CRS were isolated and identified by 16S rRNA sequencing. Two isolates were selected as representative strains based on their enzymatic spoilage potential and spoilage capability in CRS. By comparing the inhibition zones, ε-PL, NS, and TPs were selected from five conventional bio-preservatives (ε-PL, NS, TPs, grape seed extract (GSE), and rosemary extract (RE)) to prepare the mixed bio-preservative. The results showed that the minimum inhibitory concentration (MIC) of the mixed bio-preservative against Bacillus pumilus and Bacillus subtilis was 56.3 µg/mL. The growth curves and cell viability tests revealed that the mixed bio-preservative reduced the viability of both strains. The conductivity, alkaline phosphatase activity, and nucleic acid and soluble protein leakage indicated that the mixed bio-preservative disrupted the integrity of the cell walls and membranes of the two isolates in a concentration-dependent manner. Scanning electron microscopy further confirmed the damage to the cell membranes of the two isolates by the mixed bio-preservative. Overall, the mixed bio-preservative exhibited excellently inhibitory effects on the SSB and could be a promising method for the preservation of CRS.