Objective To determine if miRNA (miR) expression in umbilical cord blood and umbilical cord tissue differs between neonates with early onset sepsis (EOS) versus neonates without true infection. Methods Retrospective case-control study design of human patients with EOS (n = 8), presumed sepsis (N = 12) and non-infected control patients (N = 21). Differential expression of 300 miRs was examined using the MIHS-3001ZE-miScript miRNA PCR Array Human miFinder 384HC. Expression levels of miRs were normalized using the global Ct mean of expressed miR and compared between groups. Data analysis was performed using GeneGlobe data analysis software. Ratios of over and under-expressed miRs were calculated and compared between groups using receiver operating characteristic (ROC) curves. Results Both umbilical cord plasma and umbilical cord tissue revealed several miRs with differential expression with little overlap between the two specimen types. The most overexpressed miR in plasma of EOS patients was miR-211-5p and the most overexpressed in EOS cord tissue was miR-223-5p. ROC curves comparing the ratios of over and under-expressed miRs for EOS patients and controls resulted in an area under the curve of 0.787 for cord plasma (miR-211-5p/miR-142-3p) and 0.988 for umbilical cord tissue (miR-223-5p/miR-22-3p), indicating good discrimination. Conclusions miRs show differential expression in EOS versus non-infected controls and presumed sepsis. A ratio of over and under-expressed miRs can provide a potentially sensitive and specific diagnostic test for EOS.