Abstract: Objective: To isolate and identify Lactobacillus gasseri from 125 fecal samples of healthy infants, and to evaluate its safety and antimicrobial activity. Methods: In this study, fecal samples of 1-month-old healthy infants were taken, and the strains were separated by MRS medium, Gram staining, biochemical identification and 16S rRNA sequencing technology were used to strain identification. Subsequently, the hemolysis of the isolated L. gasseri was evaluated by blood agar. The drug sensitivity of antibiotics was determined by micro broth dilution method, and the safety of the isolated strains were evaluated. Finally, the inhibitory effects of the 15 isolated L. gasseri strains on E. coli ATCC 25922 and S. aureus ATCC 25923 were detected by Oxford cup method. Results: Fifteen strains of L. gasseri were successfully isolated from 125 fecal samples of healthy infants. Hemolysis test results showed that 14 strains of them was hemolytic negative. The 15 tested strains were highly sensitive to tetracycline, vancomycin, linezolid and rifampicin, completely sensitive to imipenem and completely resistant to trimethoprim. The results of antibacterial activity test showed that 6 strains could effectively in inhibiting both E. coli and S. aureus. Conclusion: In this study, three strains of L. gasseri were successfully isolated from healthy infant’s fecal samples, which could not only inhibit E. coli and S. aureus, but also passed the safety test (LGI 6-1, LGI 6-2 and LGI 6-3). As potential probiotic resources, they could provide a research target for further research on function exploring.