Objective: The objective of this study is to evaluate the usefulness of fecal microRNA (miR)-223 and miR-451a, as novel noninvasive biomarkers for early diagnosis of necrotizing enterocolitis (NEC) in preterm infants. Methods: Among the top-listed target miRNAs in our previous differential microarray analysis, miR-223 and miR-451a were quantified in a pilot validation case-controlled study (NEC vs. non-NEC/nonsepsis infants; n = 6 in each group). A definitive prospective cohort study (n = 218) further assessed their clinical usefulness as noninvasive and specific diagnostic biomarkers. Fecal calprotectin was quantified in parallel for comparison. Results: Of 43 proven NEC cases in the cohort study, 24 (55.8%) had fecal samples recovered within the first 3 days of clinical presentation. Fecal miRNA-223 (10.5 fold), miR-451a (4.5 fold), and calprotectin (2.1 fold) concentrations were significant higher in NEC compared with the non-NEC group (p < 0.009). Accepting a minimum sensitivity of 0.75, the positive predictive values (PPVs) ranged between 0.19 and 0.20. Combining fecal biomarkers and CRP (Day 1) could marginally increase the PPVs (0.31–0.34) but adversely lowered the sensitivity (0.54–0.63). Conclusions: Although fecal miRNA biomarkers and calprotectin concentrations were significantly higher in the NEC group, the considerable overlapping of concentrations between groups and low recovery of stool specimens within 72 h of clinical presentation rendered fecal noninvasive tests of limited clinical value in guiding diagnosis of NEC during the acute phase. A further study is underway to evaluate their roles in surveillance for predicting high-risk premature infants developing NEC.