Background Sepsis poses significant challenges in early detection and treatment due to its heterogeneous nature and overlaps with other conditions, such as pneumonia. Identifying reliable biomarkers is crucial for intervention and outcomes. Methods A total of 190 adults, aged 18 years and above, categorized into four groups: sepsis (n=50), pneumonia (n=53), pneumonia with sepsis (n=34), and healthy controls (n=53). Patients received treatments in the ICU, and the healthy were donors from blood bank. ELISA and qRT-PCR utilized to assess protein and mRNA levels in peripheral blood samples from all participants. ROC curve analyses conducted to determine diagnostic accuracy. Results mRNA and protein levels of HBD-3 and -6 significantly elevated in all patients compared to healthy. ROC analysis revealed high sensitivity but low to moderate specificity for HBD-3 and -6. HBD-6 emerged as a promising biomarker for sepsis patients with or without pneumonia. Conclusion HBD-3 mRNA and HBD-6 protein could be potential as diagnostic markers for sepsis and pneumonia. Despite limitations such as sample size and technical constraints, this study emphasizes the potential of using HBDs as biomarkers, requiring further research to enhance their reliability and clinical usefulness.