Premature birth, especially if born before week 32 of gestation, is associated with increased risk of neonatal morbidity and mortality. Prophylactic use of probiotics has been suggested to protect preterm infants via supporting a healthy gut microbiota (GM) development, but the suggested strains and doses vary between studies. In this study, we profiled the GM of 5, 10 and 30-day fecal samples from two cohorts of preterm neonates (born <30 weeks of gestation) recruited in the same neonatal intensive care unit. One cohort (n = 165) was recruited from September 2006 to January 2009 before probiotics were introduced in the clinic. The second cohort (n = 87) was recruited from May 2010 to October 2011 after introducing Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis ssp. lactis BB-12 supplementation policy. Through V3-V4 region 16S rRNA gene amplicon sequencing, a distinct increase of L. rhamnosus and B. animalis was found in the fecal samples of neonates supplemented with probiotics. During the first 30 days of life, the preterm GM went through similarly patterned progression of bacterial populations. Staphylococcus and Weissella dominated in early samples, but was gradually overtaken by Veillonella, Enterococcus and Enterobacteriaceae. Probiotic supplementation was associated with pronounced reduction of Weissella, Veillonella spp. and the opportunistic pathogen Klebsiella. Potential nosocomial pathogens Citrobacter and Chryseobacterium species also gradually phased out. In conclusion, probiotic supplementation to preterm neonates affected gut colonization by certain bacteria, but did not change the overall longitudinal bacterial progression in the neonatal period. Abbreviations: GM: Gut microbiota; ASV: Amplicon sequence variant; NEC: Necrotizing enterocolitis; DOL: Days of life; NICU: Neonatal intensive care unit; ESPGHAN: European Society for Pediatric Gastroenterology, Hepatology and Nutrition; Db-RDA: Distance-based redundancy analysis; PERMANOVA: Permutational multivariate analysis of variance; ANCOM: Analysis of compositions of microbiomes; LGG: Lacticaseibacillus (former Lactobacillus) rhamnosus GG; BB-12: Bifidobacterium animalis ssp. lactis BB-12; DGGE: Denaturing Gradient Gel Electrophoresis.