Introduction Zonulin (ZO), a new diagnostic biomarker of intestinal permeability, was tested in newborns presenting symptoms of infection and/or inflammation of the gut or being at risk of intestinal pathology. Material and Methods Serum ZO was assessed in 81 newborns diagnosed with sepsis, necrotizing enterocolitis (NEC), rotavirus infection, and gastroschisis, also in extremely low gestational age babies, and in controls (healthy newborns). ZO concentration was compared to C-reactive protein (CRP) and procalcitonin (PCT) values, leucocyte and platelet count, basic demographic data, and the value of the Neonatal Therapeutic Intervention Scoring System (NTISS). Results Median values of ZO were markedly higher in groups with rotavirus infection and gastroschisis (36.0 (1-3Q: 26.0–43.2) and 20.3 (1-3Q: 17.7–28.2) ng/ml, resp.) versus controls (3.5 (1-3Q: 2.7–4.8) ng/ml). Its concentration in the NEC group was twice as high as in controls but did not reach statistical significance. ZO levels were not related to NTISS, CRP, and PCT. Conclusions Zonulin is a promising biomarker of intestinal condition, markedly elevated in rotavirus infections. Its role in defining the severity of necrotizing enterocolitis and the risk for perforation is not well described and needs further evaluation. An increase in zonulin may not be parallel to the release of inflammatory markers, and low CRP should not exclude an injury to neonatal intestine.