Background: Neonatal diarrhoea is one of the most common diseases in lambs, causing economic losses and welfare issues. The endothelial glycocalyx (eGC) plays a crucial role in vascular integrity and inflammatory regulation. However, its involvement in neonatal lamb diarrhoea remains unclear. Objectives: This study aimed to evaluate eGC damage in neonatal lambs with diarrhoea by assessing biomarkers, including nitric oxide (NO), heparan sulphate (HS), angiopoietin-2 (Ang-2) and A disintegrin and metalloproteinase 15 (ADAM15). Methods: A total of 68 neonatal lambs with diarrhoea (39 infected with Escherichia coli and 29 with Cryptosporidium parvum) and 20 healthy controls were included. Diarrhoea aetiology was confirmed via faecal antigen tests and microscopic examination. Blood samples were collected for hemogram and biomarker analysis using sheep-specific ELISA kits. Results and Conclusions: Lambs with diarrhoea showed significantly higher total leukocyte (white blood cell [WBC]), granulocyte (GRA) and monocyte (MON) counts (p < 0.01). Serum NO concentrations were significantly elevated in lambs with diarrhoea, whereas HS and Ang-2 concentrations were lower (p < 0.01). E. coli–infected lambs had significantly higher ADAM15 concentrations than those with Cryptosporidium infection (p < 0.001). These findings suggest that NO may have a protective and/or anti-inflammatory role in neonatal lamb diarrhoea, potentially maintaining eGC integrity despite the inflammatory process.