Orofacial clefts (OFC) present different phenotypes with a postnatal challenge for oral microbiota development. In order to investigate the impact of OFC on oral microbiota, smear samples from 15 neonates with OFC and 17 neonates without OFC were collected from two oral niches (tongue, cheek) at two time points, i.e. after birth (T0: Ø3d OFC group; Ø2d control group) and 4–5 weeks later (T1: Ø32d OFC group; Ø31d control group). Subsequently, the samples were analyzed using next-generation sequencing. We detected a significant increase of alpha diversity and
anaerobic
and
Gram-negative species
from T0 to T1 in both groups. Further, we found that at T1 OFC neonates presented a significantly lower alpha diversity (lowest values for high cleft severity) and significantly higher levels of
Enterobacteriaceae (Citrobacter, Enterobacter, Escherichia-Shigella, Klebsiella), Enterococcus, Bifidobacterium, Corynebacterium, Lactocaseibacillus, Staphylococcus, Acinetobacter
and
Lawsonella
compared to controls. Notably, neonates with unilateral and bilateral cleft lip and palate (UCLP/BCLP) presented similarities in beta diversity and a mixture with skin microbiota. However, significant differences were seen in neonates with cleft palate only compared to UCLP/BCLP with higher levels of
anaerobic species
. Our findings revealed an influence of OFC as well as cleft phenotype and severity on postnatal oral microbiota maturation.