Diabetic foot ulcers develop within days of an initial foot injury and remain undetected due to neuropathy and ischaemia. Damage to the protective skin layer enables bacterial colonisation and potential development of antimicrobial tolerant biofilm infection. Identifying the microflora inhabiting chronic non-healing diabetic foot ulcers (DFU) is paramount if we are to increase our understanding of the multifaceted nature of these debilitating wounds. Obtaining both clinically and scientifically informative samples from wounds for bacterial profiling, with minimal impacts to the patients, is desirable. Tissue biopsies have been the sample of choice when using molecular techniques. However biopsies are invasive and collection requires trained clinicians. This study investigates whether swabs collected using the Levine technique provide comparable information to tissue biopsies, on the microbiome of DFU’s using 16SrRNA gene sequencing. Paired swab and tissue biopsy samples were sequenced from the ulcers of 21 diabetic patients. Organisms were classified as distinct operational taxonomic units (OTUs) based on 97% sequence homology after blasting against Greengenes reference database. There was no significant difference in the bacterial richness (number of distinct OTU’s) or Shannon’s diversity index between the paired swab and tissue biopsy samples, whereas the abundance of OTU’s was significantly higher in swabs (p<0.05). Multivariate analysis revealed significant differences in the community composition of bacteria from swabs compared to tissue biopsies. More unidentified, “uncultured” bacteria and archaea (p<0.05) were found in the tissue samples, whereas common and potential pathogens including anaerobes, were more prevalent in the swabs. Significantly higher abundances of Staphylococcus spp., Streptococcus spp., Corynebacterium spp. and Anaerococcus spp. were found in swabs compared to tissue biopsies, using Wilcoxon paired t-tests ( p<0.05). Overall, despite a lack of concordance in the bacterial communities from most paired swab and tissue samples, this study indicates that 16SrRNA gene sequencing from both swabs and tissue biopsies can provide clinically useful information from DFUs, with evidence that more potential pathogens could be detected in swabs.