[Objective] To evaluate the effect of oral administration of probiotics on the colonization of Escherichia coli K1 and Group B streptococcus (GBS) in the guts and vaginal among pregnant women.[Methods] Vaginal and rectal secretion specimens were collected from 2539 health pregnant women in Guangdong from 2011 to 2017. First, 47 healthy pregnant women involved were randomly divided into the 2 groups. The treatment group received probiotics, while the control group without. Real-time PCR was used to detect the quantitative colonization changes of E. coli K1 in the two groups thereafter. Then, 50 pregnant women subjected to the GBS screening by PCR were randomly divided into 2 groups. Later, real-time PCR was used to detect the quantitative changes of GBS in the two groups. Then, real-time PCR was used to detect GBS in the genital tract and rectal specimens for recording the colonization of GBS. [Results] With no statistical differences between the groups in term of age, multipara proportion and level of education (P>0.05), the colonization of E. coli K1 in the treatment group declined dramatically (F=32.866, P<0.001). Women in the oral probiotics group have lower level of colonization of E. coli K1 (F=41.546, P<0.001). The results suggest that oral administration of probiotics may inhibit the colonization of E. coli K1 in the intestines and vaginas of pregnant women. Meanwhile, the colonization level of GBS in probiotics group declined after taking probiotics. Finally, the GBS carrier rate of the pregnant woman was 8.07% by PCR. [Conclusion] The GBS carrier rate of the screening pregnant women in Guangdong, China was 8.07%. The results suggest that oral administration of probiotics to pregnant women can inhibit the colonization of E. coli K1 in the intestines and vaginas of these women, which may prevent neonatal bacterial meningitis in newborns as a result of hematogenous spread.