Neutrophil extracellular traps (NETs) contribute to antimicrobial defense but may promote tissue damage when produced in excess or insufficiently cleared. Acute appendicitis is characterized by neutrophil-driven inflammation, but NET-associated structures and their compartment-specific dynamics have not yet been systematically evaluated. We analyzed appendiceal tissue across histopathological stages of adult appendicitis patients (n = 60), including layer-specific assessment of citrullinated histone H3 (CitH3), myeloperoxidase (MPO), B-DNA and modified DNA. In addition, we quantified NET-associated markers in the circulation of patients with histopathologically staged appendicitis (up to n = 52) and controls without appendicitis (n = 29). CitH3- and MPO-positive deposits increased with histopathological severity, formed large patches, and showed increasing involvement of the submucosa and muscularis in advanced stages. In parallel, the DNA ratio shifted toward modified DNA conformations, while B-DNA decreased. Circulating CitH3, MPO-DNA complexes, and cfDNA increased with disease stage and distinguished acute appendicitis from non-appendicitis controls. These findings suggest that acute appendicitis is not only defined by neutrophil transmigration but also by spatial and structural remodeling of NET-associated extracellular chromatin. The shift in DNA conformational patterns most likely reflects altered extracellular chromatin processing and, consequently, differential persistence of extracellular DNA. Whether circulating NET-associated markers can support diagnosis and severity stratification of appendicitis requires further validation.