The pioneer microbiota of the neonate may affect future actions of the immune system. This study aimed to map the pioneer microbiota in healthy neonates vaginally born at term. A subgroup of neonates born large for GA (LGA) was compared with the neonates appropriate for GA (AGA). Fecal samples were collected, within 48 h after birth, from 79 neonates. Quantitative PCR was used for enumeration of Lactobacillus, a subgroup of Lactobacillus common in the vagina, Bifidobacterium, Enterococcus, Enterobacteriaceae, and the Bacteroides fragilis group. Cloning and sequencing were applied for subgroups of neonates born LGA or AGA. Lactobacillus was detected in all neonates, whereas other bacterial groups were detected only in 14 to 30% of the subjects. The prevalence of Gram-negative Proteobacteria was higher in neonates born LGA, whereas Gram-positive Firmicutes was more prevalent in neonates born AGA (p < 0.001). This study contributed to increased knowledge of the pioneer microbiota and indicates that neonates born LGA had significantly different microbiota compared with those born AGA. As the early microbiota can be important for maturation of the immune system, the outcome from this study may be relevant in the care of pregnant woman and newborns.