Diabetic foot ulcer (DFU) is one of the most serious and costly complications of diabetic patients. Enterococcus faecalis (E. faecalis) represents one of the most virulent microorganisms in diabetic foot infections (DFIs). We therefore aimed to study the frequency and precise identification of E. faecalis in DFU. Six hundred thirty specimens collected from diabetic foot patients were used in the current investigation. Biochemical identification was carried out by the Vitek® 2 system. Proteomic analysis was implemented by MALDI-TOF MS and confirmed by SYBER Green real-time polymerase chain reaction (SYBER Green qPCR). According to the results, the overall frequency of E. faecalis in patients with DFU was 8.25% (52/630). Out of 52 E. faecalis strains, 40 isolates were isolated from males and 12 from females. The results of biochemical identification revealed that 92.30% (48/52) of E. faecalis isolates were properly recognized at the species level. Whereas 100% (52/52) of E. faecalis isolates were properly recognized by MALDI-TOF MS as 44.23% (23/52), 51.92% (27/52) and 3.84% (2/52) with a score value ranging from 2.300 to 3.000, 2.000-2.299 and 1.700-1.999 Da, respectively. Seven E. faecalis virulence genes, including asa1, GelE, cylA, esp, hy1, VanA, and VanB, were detected by SYBER Green RT-PCR. In conclusion, E. faecalis was the utmost predominant single organism isolated from the DFIs. MALDI-TOF mass spectrometry is considered a fast, trustworthy and economic detection method for various significant microorganisms. E. faecalis isolates were also found to carry several virulence genes. Our findings may serve as an urgent issue for supplementary investigations of contagions caused by E. faecalis.