Background: The mannose-binding lectin (MBL) is a collectin family member that is produced by the liver as an acute-phase protein. MBL activates macrophages, improves phagocytosis, and aids in complement activation. Low serum MBL levels raise the risk of infection, particularly when combined with other conditions such as immune deficiencies of various origins. Because their adaptive immunity has not yet developed, neonates are considered immunocompromised. This study aimed to determine the role of serum MBL levels in the diagnosis of neonatal sepsis, as well as the role of MBL2 gene polymorphism at codon 54 (G/A) in the development of neonatal sepsis. Methods: This case-control study was carried out on 100 neonates classified into two groups: 50 preterm neonates diagnosed with sepsis and 50 healthy preterm neonates with no clinical signs or laboratory evidence of sepsis who were enrolled as a control group. Assessment of history, clinical examination, and investigations (complete blood count, C‑ reactive protein, blood culture, MBL2 gene polymorphism at codon 54 (G/A), MBL serum level) were performed. Results: Mean serum MBL was significantly lower in septic neonates (45.5 + 10.0 ng/mL) compared to the control group (69.1 + 26.1 ng/mL) (P<0.0001), and had a good predictive value for discrimination between septic neonates and controls with an area under the receiver-operating characteristic (ROC) curve (AUC) of 0.92 (95% CI, 0.849 to 0.965) (p-value <0.001). The normal genotype (GG) of codon 54 was significantly lower in patients (30/50, 60.0%) compared to controls (43/50, 86.0%) (P=0.006), while the heteromutant (GA) genotype of codon 54 was significantly higher in patients (17/50, 34.0%) than in controls (7/50, 14.0%) (P=0.033). The codon 54 alleles were statistically different in patients compared to controls; the G allele was significantly lower in patients (77%) than in controls (93%) (P=0.0025), while the A allele was significantly higher in patients (23%) than in controls (7%) (P=0.0025). Conclusions: The presence of the variant A allele in the MBL2 gene at codon 54 may be associated with a decreased serum level of MBL, and therefore may be associated with an increased risk of neonatal sepsis.