Diabetic foot ulcer (DFU) is becoming one of the major complications around the world with associated consequences such as lower-extremity amputation, high morbidity, mortality and hospitalization. It has the possibility to become the next global epidemic. Major part of the concern comes from the diverse bacterial and fungal population that is found at the infection site and their growing antimicrobial resistance. If the threat of antimicrobial resistance is not dealt with than it will rise to become the main cause of mortality and below knee amputation in case DFU. Also, most of the time main focus is given on detecting bacterial population which causes the fungal population to go unnoticed and act as the silent enemy. Bacterial and fungal prevalence scenario from different countries have been discussed in this study along with the alarming antibiotic resistance scenario around the globe. Furthermore, choosing the correct technique to identify them also plays a vital role. With proven lacking’s of the culture-based methods maybe it is time to move on to the faster and more specific molecular methods. As, many of the molecular techniques have already proven to be more efficient. This review discussed the bacterial and fungal prevalence along with their growing antimicrobial resistance and evaluated different biochemical and molecular techniques in identification process.