Prematurity has got special challenge for clinicians and also other medical staff, such as microbiologists. Immature host defense mechanisms support early-onset sepsis, which can be very serious with very high mortality. While the past decade has been marked by a significant decline in early-onset group B streptococcal (GBS) sepsis in both term and preterm neonates, the overall incidence of early-onset sepsis has not decreased in many centers, and several studies have found an increase in sepsis due to gram-negative organisms. With increasing survival of these more fastidious preterm infants, late-onset sepsis or specially nosocomial bloodstream infection (BSI) will continue to be a challenging complication that affects other morbidities, length of hospitalization, cost of care, and mortality rates. Especially the very low birthweight (VLBW) infants sensitive to serious systemic infection during their initial hospital stay. Sepsis caused by multiresistant organisms and Candida spp. are also increasing in incidence, has become the most common cause of death among preterm infants. This review focuses on the clinical microbiology of neonatal sepsis, particularly among preterm babies, summarizing the most frequent bacterial and fungal organisms causing perinatally acquired and also nosocomial sepsis.