Purpose Different bacteria lead to divers diabetic foot infections (DFIs), and some bacteria probably lead to higher amputation and mortality risks. We assessed mortality and amputation risk in relation to bacterial profiles in people DFI and investigated the role of sampling method. Methods We included people (> 18 years) with DFI in this retrospective study (2011–2020) at a Dutch tertiary care hospital. We retrieved cultures according to best sampling method: (1) bone biopsy; (2) ulcer bed biopsy; and (3) swab. We aggregated data into a composite determinant, consisting of unrepeated bacteria of one episode of infection, clustered into 5 profiles: (1) Streptococcus and Staphylococcus aureus; (2) coagulase-negative Staphylococcus, Cutibacterium, Corynebacterium and Enterococcus; (3) gram-negative; (4) Anaerobic; and (5) less common gram-positive bacteria. We calculated Hazard Ratio’s (HR’s) using time-dependent-Cox regression for the analyses and investigated effect modification by sampling method. Results We included 139 people, with 447 person-years follow-up and 459 episodes of infection. Sampling method modified the association between bacterial profiles and amputation for profile 2. HR’s (95% CI’s) for amputation for bacterial profiles 1–5: 0.7 (0.39–1.1); stratified analysis for profile 2: bone biopsy 0.84 (0.26–2.7), ulcer bed biopsy 0.89 (0.34–2.3), swab 5.9*(2.9–11.8); 1.3 (0.78–2.1); 1.6 (0.91–2.6); 1.6 (0.58–4.5). HR’s (95% CI’s) for mortality for bacterial profiles 1–5: 0.89 (0.49–1.6); 0.73 (0.38–1.4); 2.6*(1.4–4.8); 1.1(0.58–2.2); 0.80(0.19–3.3). Conclusions In people with DFI, there was no association between bacterial profiles in ulcer bed and bone biopsies and amputation. Only in swab cultures, low-pathogenic bacteria (profile 2), were associated with a higher amputation risk. Infection with gram-negative bacteria was associated with a higher mortality risk. This study underlined the possible negative outcome of DFI treatment based on swabs cultures.