The aim of this work is to demonstrate how current molecular techniques should be integrated in the diagnostic process and can have a crucial role in the management of oral fungal infections. A case of median rhomboid glossitis Candida-associated and its resolution will be described step by step. At the time of the first observation, the lesion on the surface of the tongue did not respond to the previous administration of topical antifungal agent, such a nystatin. Firstly, in order to identify the causative agent and confirm Candida albicans infection, a brushing of the lesion was performed and polymerase chain reaction analysis was carried out. In addition, deoxyribonucleic acid sequencing method, known as Pyrosequencing(®), was used in the detection of point mutations commonly associated with fluconazole resistance and consequently, in the prediction of susceptibility to azole agents. According to molecular findings, the administration of fluconazole has therefore led to resolution of the case in 2 weeks. This case highlights how the use of molecular techniques, now-a-days, can assist the clinicians to quickly obtain the report with highly accurate and precise results and appropriately support them in the diagnosis and therapeutic process.