The diagnosis of neonatal invasive fungal disease (IFD) is difficult and often delayed. The platelet parameters and (1, 3)-β-D-Glucan (BG) may be useful for diagnosing IFD, but their diagnostic performance are not well characterized in neonates. We studied 63 preterm infants with IFD, 160 preterm infants without sepsis (preterm control), and 41 preterm infants with bacterial sepsis. Platelet parameters at the first day of onset of IFD and at the fourteenth day after antifungal treatment were evaluated. Serum BG was measured. Platelet count (PC), plateletcrit (PCT), and platelet distribution width (PDW) values were significantly lower, and mean platelet volume (MPV) values significantly higher in the IFD versus preterm control infants. PC and PCT values were much lower in infants with IFD versus bacterial sepsis, and there were significant differences in BG value between the two groups. After 14 days of antifungal treatment, significant elevations in PC, PCT, PDW and reductions in MPV levels in IFD group were observed. Receiver operating characteristic (ROC) curves showed that PC and PCT were strong predictors of IFD. The PC and PCT cut-offs for predicting IFD were 119.5 (sensitivity 78%, specificity 95%) and 0.21 (sensitivity 83%, specificity 85%), respectively. There were significant differences in PC and PCT levels between deceased and survived patients. The PC and PCT cut-offs for predicting deceased IFD were 39 (sensitivity 62%, specificity 86%) and 0.04 (sensitivity 50%, specificity 95%), respectively. The sensitivity in diagnosing IFD by a BG cutoff of ≥10 pg/ml was 68.3% and specificity was 75.6%. PC and PCT levels in the BG ≥400 pg/ml group were significantly lower compared to the BG<400 pg/ml group. Platelet parameters and BG could be useful biomarkers for the diagnosis and prognosis of neonatal IFD.