The main goal of the study was to evaluate the usefulness of the culturomics approach in the reflection of diabetic foot infections (DFIs) microbial compositions in Poland. Superficial swab samples of 16 diabetic foot infection patients (Provincial Polyclinical Hospital in Toruń, Poland) were subjected to culturing using 10 different types of media followed by the identification via the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Biotyper platform. Identified 204 bacterial isolates representing 18 different species—mostly Enterococcus faecalis (63%) and Staphylococcus aureus (44%). Most of the infections (81%) demonstrated a polymicrobial character. Great differences in the species coverage, the number of isolated Gram-positive and Gram-negative bacteria, and the efficiency of the microbial composition reflection between the investigated media were revealed. The use of commonly recommended blood agar allowed to reveal only 53% of the entire microbial composition of the diabetic foot infection samples, which considerably improved when the chromagar orientation and vancomycin-resistant enterococi agar were applied. In general, efficiency increased in the following order: selective < universal < enriched < differential media. Performed analysis also revealed the impact of the culture media composition on the molecular profiles of some bacterial species, such as Corynebacterium striatum, Proteus mirabilis or Morganella morganii that contributed to the differences in the identification quality. Our results indicated that the culturomics approach can significantly improve the accuracy of the reflection of the diabetic foot infections microbial compositions as long as an appropriate media set is selected. The chromagar orientation and vancomycin-resistant enterococi agar media which were used for the first time to study diabetic foot infection microbial profiles demonstrate the highest utility in the culturomics approach and should be included in further studies directed to find a faster and more reliable diabetic foot infection diagnostic tool.