Aim: This study aimed to evaluate the role of preventive strategies in reducing neonatal sepsis by analyzing microbial and biochemical risk factors in a pediatric population. Materials and Methods: A prospective observational study was conducted over one year, involving 100 neonates admitted to the NICU of a tertiary care hospital. Neonates were categorized into two groups based on preventive care exposure: the Adequate Prevention Group and the Inadequate Prevention Group. Data included demographic characteristics, preventive measures, laboratory investigations (biochemical markers such as CRP and procalcitonin), and microbiological analyses. Outcomes included sepsis incidence, clinical severity, and mortality rates. Results: The Adequate Prevention Group had significantly better outcomes, including higher birth weights (2.85 ± 0.45 kg vs. 2.45 ± 0.50 kg, p = 0.002), longer gestational ages (38.20 ± 1.50 weeks vs. 36.80 ± 2.00 weeks, p = 0.001), and improved Apgar scores (8.50 ± 1.20 vs. 7.80 ± 1.50, p = 0.005). Sepsis incidence was significantly lower in this group (20.00% vs. 45.00%, p = 0.008), with Escherichia coli being the most common pathogen. Biochemical markers (CRP, procalcitonin, and lactate) were significantly elevated in the Inadequate Prevention Group, correlating with greater disease severity. Clinical outcomes were also superior in the Adequate Prevention Group, with reduced mortality (6.67% vs. 25.00%, p = 0.011), shorter NICU stays (12.50 ± 3.80 days vs. 18.20 ± 4.50 days, p = 0.001), and lower mechanical ventilation requirements (16.67% vs. 40.00%, p = 0.005). Conclusion: Preventive strategies such as antenatal care, hygienic delivery practices, early breastfeeding, and umbilical cord care significantly reduce neonatal sepsis incidence and severity. Elevated CRP and procalcitonin levels remain critical markers for early diagnosis and management. A multifaceted approach combining preventive measures and robust diagnostic protocols is essential for improving neonatal outcomes and reducing healthcare burdens.