The incidence of spontaneous intestinal perforation (SIP) increases up to 10% with decreasing gestational age (GA). We aimed to explore early biomarkers for predicting SIP in preterm infants. In this case–control study, neonates born at ≤34 weeks GA diagnosed with SIP were compared with GA and/or birth-weight-matched neonates diagnosed with necrotizing enterocolitis (NEC). Laboratory markers assessed prior and adjacent to the day of SIP or NEC diagnosis were evaluated. The cohort included 16 SIP and 16 matched NEC infants. Hyperlactatemia was less frequent in SIP than in NEC infants (12% vs. 50%,p= 0.02). The platelets count was lower in SIP than in NEC infants (p< 0.001). Glucose levels strongly correlated with lactate levels (p= 0.01) only in the NEC group. The odds of being diagnosed with SIP decreased as lactate levels increased (OR = 0.607, 95% CI: 0.377–0.978,p= 0.04). Our results suggest that a combination of laboratory markers, namely glucose and lactate, could help differentiate SIP from NEC at early stages so that, in the presence of an elevated blood glucose, an increase in blood lactate was associated with a decrease in the odds of being diagnosed with SIP.