Numerous studies have reported bacterial DNA in first‐pass meconium samples, suggesting that the human gut microbiome is seeded prior to birth. However, these studies have not been able to discriminate between DNA from living bacterial cells, DNA from dead bacterial cells or cell‐free DNA. Here we have used propidium monoazide (PMA) together with 16S rRNA gene sequencing to determine whether there are intact bacterial cells in the fetal gut. DNA was extracted from first‐pass meconium (n = 5) and subjected to 16S rRNA gene sequencing with/without PMA treatment. All meconium samples, regardless of PMA treatment, contained detectable levels of bacterial DNA; however, treatment with PMA prior to DNA extraction decreased the DNA yield by approximately 20%. PMA‐treated meconium samples did not differ significantly from untreated samples in terms of observed number of OTUs (P = 0·945); although they did differ taxonomically, with around one quarter of OTUs identified in untreated samples only, suggesting that they have originated from cell‐free/nonviable DNA. The mean Sørensen coefficient for treated vs untreated samples was 0·527. Our findings suggest that the fetal gut is seeded with intact bacterial cells prior to birth. This is an important finding, as exposure to live bacteria during gestation might have a significant impact on the developing fetus.