Diabetic foot ulcers (DFUs) are common complications for diabetic patients, often exacerbated by complex polymicrobial biofilm infections. While the majority of DFU studies are bacterial focused, fungi have also been identified. This study aims to investigate the prevalence of fungi in DFUs, as well as their potential role and influence on persistence and wound healing. Consecutive DFU swabs were collected from 128 patients (n = 349). Fungal positivity was assessed using enhanced culture and real-time qPCR. Routine microbiology cultures were carried out as part of standard care in the clinics, and their results were then compared to our laboratory investigation. Routine and enhanced culture resulted in similar rates of fungal detection (~9%), whereas qPCR resulted in a higher rate of detection (31%). Notably, the predominant yeast Candida parapsilosis was present in ischaemic and penetrating bone wounds. These findings support existing evidence of fungal presence in DFUs. We demonstrated that routine diagnostic methods are sufficient for fungal detection, but enhanced culture methods allow for more precise fungal identification. Finally, while fungal presence does not appear to impact patient outcomes in our study, their role within these infections remains poorly understood, and further studies are needed to fully understand their relationship to the microbiome.