Fracture non-union represents a complex clinical challenge resulting from an incompletely understood interplay between mechanical and biological factors. Infection frequently contributes to non-union but many cases are misdiagnosed due to a lack of classical clinical symptoms. This study characterized peripheral blood mononuclear cells (PBMCs) from aseptic non-union (NU-AS, n = 24) and fracture-related infected non-union (NU-FRI, n = 20), and compare them to healed controls (H, n = 18). High-dimensional mass cytometry (CyTOF) revealed significant elevations of regulatory T cells (Tregs; p = 0.0028) and T helper 1 (Th1) cells (p = 0.0073), and reduced expression of the activation marker CD38 in CD4+ T cells (p = 0.0016) and Tregs (p = 0.0017) in NU compared to H. In a subgroup analysis between NU-AS and NU-FRI, monocyte and CD38+ Treg cell counts provided excellent diagnostic potential, with the combination achieving a sensitivity of 100% and a specificity of 91.7%. These findings highlight an important role of the activation marker CD38 in diagnosing chronic subclinical infection, which promises earlier identification of appropriate management of these patients.