Introduction. Bacterial biofilm in wounds prevents healing by acting as a physical barrier to wound closure and hyperactivating local inflammatory processes, thus making its removal a high priority. The authors previously have shown that adding topical oxygen to standard wound care increased healing of Texas Grade II and III diabetic foot ulcers (DFUs), which they hypothesized was a result of alterations of the wound microbiome/biofilm. Objective. This study aims to determine the mechanism of action of topical oxygen in DFUs by examining the diversity of bacterial genera present in DFUs treated with topical oxygen. Materials and Methods. Six patients with chronic DFUs had their wounds swabbed weekly over an 8-week period of continuous topical oxygen treatment, and microbiome diversity was assessed by metagenomic 16S rDNA sequencing using a next-generation sequencing platform. Results. The wound microbiome shifted toward a diverse flora dominated by aerobes and facultative anaerobes with oxygen therapy in 5 healed wounds. In contrast, anaerobic flora persisted in a single nonhealing ulcer in the present study cohort. Conclusions. Although the sample size was small, this study suggests topical oxygen therapy may have the ability to encourage the growth of aerobic members of the wound microbiome and be an effective alternative to antibiotics in this area.