The objective of the present study is to screen and identify potential antagonistic bacteria against aquatic pathogenic Citrobacter freundii, and to evaluate its safety. Using crab-pathogenic C. freundii C1 as the indicator and the diameter of inhibition zones as the evaluation index, the good Bacillus isolate with antagonistic activity was screened, and was identified by phenotypic identification and 16S rRNA gene analysis. The safety of the isolated antagonist was assessed by assaying virulence gene, antibiotic resistance, inhibitory effect on Chlorella sp, and pathogenicity for zebrafish and Chinese mitten crabs. Isolate PNB3 with good antagonistic activity was screened from the aquaculture sediment which reached 99.99% after co-culture with C. freundii C1 for 120 h at 1.0×10⁵ CFU/mL, and was identified as Bacillus licheniformis based on phenotypic characterization and 16S rRNA gene analysis. It did not contain virulence genes including haemolysin BL gene, enterotoxin Nhe gene, enterotoxin T gene, enterotoxin FM gene, cytotoxin K gene, vomiting gene, multiple regulatory factors, and exhibited high sensitivity to compound sulfamethoxazole, ampicillin, norfloxacin, kanamycin, polymyxin B, florfenicol, gentamycin, enoxacin, cefradine, penicillin, and displayed a growth-promoting effect on Chlorella sp., and showed no pathogenicity for zebrafish and Chinese mitten crabs. In conclusion, B. licheniformis PNB3 is a potential safe antagonist against C. freundii, and has the safe potential in the biocontrol of C. freundii infection in Chinese mitten crabs.