Background: Excessive neutrophil inflammation is the hallmark of cystic fibrosis (CF) airway disease. Novel technologies for characterizing neutrophil dysfunction may provide insight into the nature of these abnormalities, revealing a greater mechanistic understanding and new avenues for CF therapies that tar- get these mechanisms. Methods: Blood was collected from individuals with CF in the outpatient clinic, CF individuals hospital- ized for a pulmonary exacerbation, and non-CF controls. Using microfluidic assays and advanced imaging technologies, we characterized 1) spontaneous neutrophil migration using microfluidic motility mazes, 2) neutrophil migration to and phagocytosis of Staphylococcal aureus particles in a microfluidic arena, 3) neutrophil swarming on Candida albicans clusters, and 4) Pseudomonas aeruginosa -induced neutrophil transepithelial migration using micro-optical coherence technology (μOCT). Results: Participants included 44 individuals: 16 Outpatient CF, 13 Hospitalized CF, and 15 Non-CF indi- viduals. While no differences were seen with spontaneous migration, CF neutrophils migrated towards S. aureus particles more quickly than non-CF neutrophils (p < 0.05). CF neutrophils, especially Hospitalized CF neutrophils, generated significantly larger aggregates around S. aureus particles over time. Hospitalized CF neutrophils were more likely to have dysfunctional swarming (p < 0.01) and less efficient clearing of C. albicans (p < 0.0 0 01). When comparing trans-epithelial migration towards Pseudomonas aeruginosa ep- ithelial infection, Outpatient CF neutrophils displayed an increase in the magnitude of transmigration and adherence to the epithelium (p < 0.05). Conclusions: Advanced technologies for characterizing CF neutrophil function reveal significantly altered migratory responses, cell-to-cell clustering, and microbe containment. Future investigations will probe mechanistic basis for abnormal responses in CF to identify potential avenues for novel anti-inflammatory therapeutics.