The diagnosis of neonatal bacterial infection remains one of the greatest and most tantalizing challenges to neonatologists. At birth it must be based on the history of pregnancy and take into account a number of now well-defined risk factors. In addition, if promptly started, antibiotic therapy can reduce its sequelae and improve the prognosis. However, the number of tests that obstetricians can rely on for the diagnosis of infection is quite limited. Tests of maternal inflammation indicators have a low specificity, culture tests are not immune from the risk of contamination, and the measurement of interleukins in the amniotic fluid and maternal blood serum is not yet routine. Observation of clinical signs therefore remains crucial to neonatologists, at the same time that new and more sophisticated laboratory tests enable them to establish a diagnosis of infection at an increasingly earlier stage. In recent years, several infection markers have been investigated, such as procalcitonin and especially C-reactive protein (CRP). Currently, the measurement of plasma concentrations of interleukins (IL), IL-6 and IL-8 in particular, appears to be one of the most sensitive and specific infection indicators in newborns. Cytokine levels are increased even before infants develop any clinical symptoms and routine laboratory tests turn positive. However, owing to their short half-life, their sensitivity decreases after 12-24 h from the onset of inflammation, increasing the risk of false negatives. Ideally, they should then be used in combination with other inflammation indicators, such as CRP. The measurement of cytokines and other new inflammatory markers might be helpful in the early diagnosis of both early-onset infection (assay in umbilical cord blood) and late-onset infection (serial assays performed during the stay in the neonatal intensive care unit). In spite of their time-consuming techniques, culture tests remain of the utmost importance to plan a targeted treatment; blood culture, in particular, is crucial to the diagnosis of sepsis.