Clinical care pathways (CPs) integrate best evidence into the local care delivery context to promote efficiency and patient safety. However, the impact of CPs on diagnostic performance remains poorly understood. The objectives of this study were to evaluate adherence to a musculoskeletal infection (MSKI) diagnostic CP and identify recurrent failure points leading to missed diagnostic opportunities (MDOs). Retrospective chart review was performed from January 2018 to February 2022 for children 6 months to 18 years of age who had an unplanned admission for MSKI after being evaluated and discharged from the pediatric emergency department (PED) for related complaints within the previous 10 days. MDOs were identified using the Revised Safer Dx. Demographic and clinical characteristics of children with and without MDOs were compared using bivariate descriptive statistics. An improvement team reviewed the diagnostic trajectories of MDOs for deviations from the MSKI CP and developed a fishbone diagram to describe contributing factors to CP deviations. The study identified 21 children with and 13 children without MSKI-associated MDOs. Children with MDOs were more likely to have an initial C-reactive protein value > 2 mg/dL (90.0% vs. 0%, p = 0.01) and returned to care earlier than children without MDOs (median 2.8 days vs. 6.7 days, p = 0.004). Factors contributing to MDOs included failure to obtain screening laboratory tests, misinterpretation of laboratory values, failure to obtain orthopedic consultation, and failure to obtain definitive imaging. Several recurrent deviations from an MSKI diagnostic CP were found to be associated with MDOs. Future quality improvement efforts to improve adherence to this MSKI CP may prevent MDOs.