Objective: To evaluate the diagnostic performance of magnetic resonance imaging (MRI) for diabetic foot osteomyelitis (DFO) and to examine the feasibility and turnaround advantage of targeted resistance-gene polymerase chain reaction (PCR) as an adjunct to conventional microbiology. Methods: This prospective single-center pilot study included 80 consecutive adults with type 2 diabetes and suspected deep foot infection or osteomyelitis. All participants underwent standardized 1.5-T MRI and reference-standard classification based on histopathology when available, with bone culture and blinded multidisciplinary adjudication used as supportive or alternative evidence when histology was unavailable or nondiagnostic. Targeted PCR assessed mecA, blaCTX-M, blaTEM, blaSHV, blaKPC, blaNDM, and blaOXA-48. MRI diagnostic estimates were calculated from a patient-level 2 × 2 table and reported with 95% confidence intervals. PCR-specific analyses used available valid molecular results, and 90-day clinical outcomes were descriptive. Results: DFO was confirmed in 46/80 participants (57.5%). MRI yielded 91.3% sensitivity (95% CI, 79.2–97.6), 82.4% specificity (65.5–93.2), 87.5% positive predictive value, 87.5% negative predictive value, and 87.5% accuracy. The positive and negative likelihood ratios were 5.17 and 0.11, respectively. Conventional culture recovered pathogens in 38/46 confirmed DFO cases (82.6%) and required 62.4 ± 9.3 hours. Valid PCR results were available for 60 participants; predefined resistance determinants were detected in 28/60 (46.7%), most frequently mecA, blaCTX-M, and blaNDM. Molecular and corresponding phenotypic resistance results showed 85.0% agreement (Cohen’s κ = 0.71). PCR results were available in approximately 5 hours, about 57.4 hours earlier than final culture on average. Antimicrobial modification after PCR availability and before the final culture report occurred in 22/60 tested participants.