Preterm infants encounter an unnatural beginning to life, with housing in neonatal intensive care units (NICUs) where they are exposed to antibiotics. Although the effectiveness of antibiotics in infection control is well established, the short- and long-term unintended effects on the microbiota of preterm infants receiving antibiotic treatment are yet to be quantified. Our aim was to investigate the unintended consequences of NICU antibiotics on preterm infants’ gut microbiota. We searched three electronic databases—Embase, PubMed, and Scopus—for records from 2010 to October 2022. Eligibility criteria included intervention and observational studies that collected stool samples and analyzed microbiota data on the effect of antibiotics on the gut microbiota of preterm infants using 16S rRNA sequencing. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed, and the quality of the studies was judged using the Cochrane Collaboration Tool for assessing risk of bias (RoB2) for clinical trials, while non-randomized studies were assessed using the Newcastle–Ottawa Scale (NOS). The initial searches yielded 7,605 papers, of which 21 were included in the review. The selected studies examined 3,669 stool samples that were collected longitudinally from 878 preterm infants in seven different countries. Preterm infants exposed to antibiotics had a reduced bacterial diversity, an increased relative abundance of pathogenic bacteria such as Enterobacteriaceae, and a decrease or absence of symbiotic bacteria such as Bifidobacterium spp., which have been shown to assist in immunity development. Antibiotic discontinuation restored diversity, with variances linked to the antibiotic spectrum and treatment duration in some but not all cases. Breastfeeding confounded the association between antibiotic use and dysbiosis. Intriguingly, the reduction of γ-aminobutyric acid (GABA), a crucial neurotransmitter for early brain development, was linked to the depletion of Veillonella spp. Despite the apparent benefits of using antibiotics on preterm infants, we conclude that they should be used only when absolutely necessary and for a short period of time. Mothers’ milk is recommended to hasten the restoration of disrupted microbiota.