Succession of gut microbial community structure for newborns is highly influenced by early life factors. Many preterm infants cared for in the NICU are exposed to parent–infant separation, stress, and pain from medical care procedures. The purpose of the study was to investigate the impact of early life stress on the trajectory of gut microbial structure. Stool samples from very preterm infants were collected weekly for 6 weeks. NICU stress exposure data were collected daily for 6 weeks. V4 region of the 16S rRNA gene was amplified by PCR and sequenced. Zero‐inflated beta regression model with random effects was used to assess the impact of stress on gut microbiome trajectories. Week of sampling was significant for Escherichia, Staphylococcus, Enterococcus, Bifidobacterium, Proteus, Streptococcus, Clostridium butyricum, and Clostridium perfringens. Antibiotic usage was significant for Proteus, Citrobacter, and C. perfringens. Gender was significant for Proteus. Stress exposure occurring 1 and 2 weeks prior to sampling had a significant effect on Proteus and Veillonella. NICU stress exposure had a significant effect on Proteus and Veillonella. An overall dominance of Gammaproteobacteria was found. Findings suggest early life NICU stress may significantly influence the developing gut microbiome, which is important to NICU practice and future microbiome research.