Background: Amniotic fluid in the uterus is beneficial for the fetus growth and protection due to its nutritional elements as well as its antibacterial and anti-inflammatory properties. Today, body membranes are increasingly being used in multiple fields. The purpose of the current study was evaluation of the antibacterial effects of amniotic fluid and comparison of its effects on pathogenic and probiotic bacteria. Methods: This experimental study was conducted on amniotic fluid obtained from 43 healthy mothers who gave birth by selective cesarean section. Then, antibacterial effects of amniotic fluids were investigated on 8 standard bacterial strains, including Bacillus cereus, Escherichia coli, Staphylococcus aureus, Shigella flexneri, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus cereus, and Lactobacillus plantarum by agar well-diffusion method. Data analysis was performed by SPSS software, vs. 22 (IBM, US). Results: Amniotic fluid revealed an inhibitory effect on the growth of bacterial strains. Staphylococcus aureus and Streptococcus pyogenes strains showed growth inhibition in 39% and 17% of samples, respectively. In other bacterial strains, there was growth inhibition in less than 5% of the samples. Also, the zone of growth inhibition for Staphylococcus aureus and Streptococcus pyogenes was significantly higher than the other strains. Amniotic fluid samples had an antibacterial effect on all pathogen strains in general, but not on the Lactobacillus plantarum probiotic strain. Conclusion: Our findings suggest that the antibacterial effect of amniotic fluid on pathogenic bacteria is significantly higher than the Lactobacillus plantarum as a probiotic one. Overall, the findings support the use of natural substances as alternative therapeutic agents to combat antibiotic resistance.