This study aimed to investigate the association between active physical comforting strategies during bedtime and autonomy strategies with the relative abundances of Veillonella, Corynebacteria, Prevotella, and Faecalibacterium genera in the gut microbiota of three-month-old infants. We also assessed the impact of mode of delivery (vaginal delivery vs. cesarean section), feeding mode (breastfeeding vs. formula feeding), and sleep patterns on the relative abundances of prespecified bacterial genera. The study also aimed to explore how different nighttime parental interventions influenced gut microbiota composition and identify potential interventions to improve health outcomes in infant growth development and sleep.