Many pediatric patients with severe bacterial infection (SBI) present with a compromised general clinical picture. Early identification of the infectious and inflammatory state can improve clinical outcomes. Although microbiological culture remains the gold standard for diagnosis, its delayed results highlight the need for early biomarkers to improve clinical decision-making and patient outcomes. During infection, the acute phase response alters lipid metabolism, compromising cholesterol efflux. In this study, we characterized the serum lipid profile and HDL subclasses in 63 children with SBI at hospital admission (T0) and discharge (T1), comparing them with 63 age- and sex-matched healthy subjects. At T0, we found a significant reduction in serum total cholesterol, LDL cholesterol, and HDL cholesterol in subjects with SBI compared to controls. Interestingly, at a cut-off value of 41.5 mg/dL, HDL cholesterol discriminated the two groups with a sensitivity of 76% and a specificity of 86% for SBI. At T1, total and LDL cholesterol increased, while HDL cholesterol remained unchanged. Moreover, we observed at T1 a clear remodeling of HDL classes, with a significant decrease in large HDL with pro-inflammatory activity and an increase in the intermediate and small HDL with anti-inflammatory activity, compared to time T0. This study proposes serum HDL cholesterol as a candidate biomarker and highlights the role of HDL cholesterol and its classes in inflammatory and immune regulation in systemic bacterial infection, potentially identifying patients who could benefit from HDL-based therapies.