Introduction: Neonatal sepsis is a severe and life-threatening condition caused by pathogens in the systemic circulation within the first 28 days of life. The classical definition of neonatal sepsis implies positive central cultures, but recent findings discuss culture-negative sepsis (clinical sepsis associated with laboratory findings). Since infected neonates initially express few non-specific clinical signs and there are unreliable biochemical markers to identify sepsis in the early stages, it is essential to improve the accuracy of diagnosis and reduce unnecessary antibiotic exposure. Objective: Our study aims to assess the influence of risk factors and the utility of currently used biomarkers in culture-negative neonatal early-onset sepsis (CN-EOS). Materials and methods: We performed a retrospective study at Bucharest University Hospital, which included 131 preterm and term newborns at risk for EOS admitted in the Neonatal Intensive Care Unit (NICU) over 12 months. The neonates included were classified into two groups: confirmed negative-culture early-onset sepsis (CN-EOS) and suspected early-onset sepsis (S-EOS). Patients from both groups received antibiotic therapy from the first day of life; the type and duration of antibiotic therapy were different in the two groups. For all the patients, we measured C-reactive protein (CRP), procalcitonin (PCT) and white blood count (WBC) at birth and after 72 h, tested blood culture in the first 24 h of life and correlated the results with clinical signs and prenatal risk factors. Categorical variables were presented as frequencies and percentages, while the continuous variables were the mean and the standard deviation. The differences between the continuous variable groups were determined by Student’s t-test or the Mann–Whitney U test, whereas for the categorical variables, the Chi-square test (X²) was employed. The performance of laboratory biomarkers (CRP and PCT) in diagnosing confirmed EOS was calculated. All the tests were statistically significant at a p-value < 0.05. Results: The findings support the significance of low birth weight and gestational age and low Apgar scores as potential indicators for EOS; PROM diagnosed with chorioamnionitis and smoking during the pregnancy were also important predictive risk factors. Respiratory signs, such as apnea and respiratory distress syndrome, were most encountered in the clinical evaluation of infants with CN-EOS. Inflammatory markers were inconsistent in CN-EOS cases, proving that they are not reliable enough for initiating, continuing or stopping antibiotic therapy. Conclusions: Culture-negative neonatal sepsis remains a significant challenge for the neonatologist, since the time elapsed between the moment sepsis is suspected and the initiation of empirical therapy can make the difference between survival and death. Continued efforts are needed to develop more reliable and effective diagnostic tools for timely and appropriate intervention.