Diabetic foot infections are a major cause of hospitalization, and delayed treatment can lead to numerous complications. The aim of this research was to investigate high-resolution spectroscopy of the wound center and periwound area for real-time estimation of multispectral signature of bacteria at the base of diabetic foot ulcers. We investigated the spectrum of the reflected visual light from diabetic foot ulcers and developed a method that identifies the presence of bacteria in the wound infections. We undertook a prospective pilot study on 18 patients with type 1 and type 2 diabetes and chronic diabetic foot ulcers. The spectral coefficients were directly compared with the results from the wound swab. The results of the multispectral analysis demonstrated 100% sensitivity, with 100% negative predictive values of identifying the presence of the bacteria, which was the cause of the infection in the wound. The results of our study suggest that the changes in the multispectral properties of the wound can be used to identify the presence of bacteria in the infected area using a noninvasive device without any contact with the wound. This technique holds great promise for real-time objective evaluation of the wound infection status beyond the standard visual assessment of diabetic foot ulcers.