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Prematurity exposes newborns to increased risks of infections and it is associated with critical morbidities. Preterm infants often require antibiotic therapies that can affect the correct establishment of gut microbiota. The aim of this study was to investigate targeted intestinal bacteria in preterm neonates with common morbidities and receiving antibiotic treatments of variable duration. Stool samples were collected after birth, at 15, 30 and 90 days of life. qPCR quantification of selected microbial groups (<i>Bifidobacterium</i> spp., <i>Bacteroides fragilis</i> group, Enterobacteriaceae, <i>Clostridium</i> cluster I and total bacteria) was performed and correlation between their levels, the duration of antibiotic treatment and different clinical conditions was studied. An increasing trend over time was observed for all microbial groups, especially for <i>Bifdobacterium</i> spp. Prolonged exposure to antibiotics in the first weeks of life affected <i>Clostridium</i> and <i>B. fragilis</i> levels, but these changes no longer persisted at 90 days of life. Variations of bacterial counts were associated with the length of hospital stay, feeding and mechanical ventilation. Late-onset sepsis and patent ductus arteriosus reduced the counts of <i>Bifidobacterium</i>, whereas <i>B. fragilis</i> was influenced by compromised respiratory conditions. This study can be a start point for the identification of microbial biomarkers associated with some common morbidities and tailored strategies for a healthy microbial development.