Background: This study aimed to evaluate the diagnostic utilities of monocyte HLA-DR as an infection marker in the identification of early-onset clinical infection and pneumonia in newborn infants. Methods: Term newborns in whom infection was suspected when they were <72 h of age were eligible for enrollment in the study. C-reactive protein (CRP), monocyte HLA-DR and neutrophil CD64 expressions were quantitatively measured at the time of sepsis evaluation (0 h) and 24 h afterwards by flow cytometry and standard laboratory method. Results: A total of 288 infants with suspected sepsis were investigated, and 93 were found to be clinically infected. There were no significant differences in monocyte HLA-DR expression between the infected, non-infected and control groups at 0 h (median (interquartile range): 13,986 (10,994–18,544), 14,234 (12,045–17,474) and 18,441 (14,250–21,537) antibody phycoerythrin (PE) molecules bound/cell), and between infected and non-infected infants at 24 h (median (interquartile range): 17,772 (12,933–25,167) and 19,406 (14,885–24,225) antibody PE molecules bound/cell). The areas under the receiver operating characteristics (ROC) curves for HLA-DR, CD64 and CRP were 0.52–0.54, 0.88–0.94 and 0.75–0.77, respectively. We were unable to determine an optimal cutoff value for HLA-DR, as the diagnostic utilities of any cutoff point on the ROC curves were unable to satisfy the criteria (i.e. sensitivity and specificity ≧80%) for consideration as an useful diagnostic marker of infection. Conclusions: Our findings did not support the use of monocyte HLA-DR alone or in combination with other infection markers in the diagnosis of early-onset clinical infection and pneumonia in term newborns.