Background Optimal timing of bone culture obtainment relative to antibiotic initiation in diabetic foot osteomyelitis is unknown. This study aims to determine whether antibiotic administration prior to bone sample collection affects microbiological yield. Methods This was a single-center retrospective study. Patients at least 18 years old admitted for diabetic foot osteomyelitis management were included. Patients included in the study had an evaluable bone sample and met the following criteria: presence of imaging demonstrating osteomyelitis, documentation of wound probing to bone, or visible bone in wound; plus an elevated erythrocyte sedimentation rate or elevated C-reactive protein. Patients included also received at least one dose of antibiotics during the hospitalization. Patients with necrotizing fasciitis and those who left against medical advice were excluded. Results One hundred and fifteen patients were included; 78 received antibiotics prior to bone sample obtainment (prior group), and 37 received antibiotics post sample obtainment (post group). Average time from admission to bone sample obtainment in the prior group was 1.8 ± 2.1 days and 1.1 ± 1.0 days in the post group. There was a non-significant difference in microbiological yields between the two groups (78.2% vs 75.7%, p = 0.762). Further examining the prior group, the average time between the antibiotic initiation and bone sample obtainment was not significantly different between those who had microbiological growth versus those who did not (2 ± 1.4 versus 1.6 ± 2.3 days [p = 0.0942]). Logistic regression analysis did not show association of microbiological yield with any of the following variables: peripheral vascular disease, end-stage renal disease, biopsy type (bedside vs open), type of osteomyelitis (acute vs chronic vs acute-on-chronic), antibiotic use prior to admission, or types of antibiotics administered. Conclusion Our data suggests that approximately two days of antibiotics prior to bone biopsy does not affect microbiological yield on cultures. Bone samples were obtained expeditiously within our cohort, which may account for the lack of difference seen. Further studies are needed to better define the duration of antibiotic therapy at which a difference in bone culture yields will be expected. Disclosures Asia N. Quan, PharmD, BCPS, BCCCP, Shionogi: Honoraria|Trevena: Honoraria Carlos Hartmann, MD, Viiv Pharmaceuticals: Speaker.