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Abstract Background Oral candidiasis is a common problem associated with head and neck radiation therapy (RT) consequences being pain, burning sensation, taste change, and systemic infection. There are difficulties in differentiating between oral candidiasis and radiation induced oral mucositis. We conducted a prospective study to explore the incidence of clinical oral candidiasis and evaluate the accuracy of diagnosis among head and neck cancer (HNC) patients receiving RT or concurrent chemoradiotherapy (CCRT). Methods This study included 86 HNC patients who had no clinical signs or symptoms of oral candidiasis. Oral mucosa and tongue swabs were carried out and analyzed three times by fungal cultures at the following time points: (1) before RT, (2) at the time of clinically diagnosed candidiasis or during RT at between the 15th–17th fraction (whichever occurred first), and (3) at the end of RT. The accuracy of clinical oral candidiasis was explored and confirmed by fungal colonization techniques. The incidence and risk factors associated with fungal colonization before and throughout the treatment were analyzed. Results The overall incidence of clinical oral candidiasis was 53.5% throughout the course of RT. Confirmation of fungal colonization was found in 39.5%, 65.9%, and 57.7% of cases before RT, during RT, and end of RT, respectively. The accuracy of the diagnosis using only clinical presentation was demonstrated in 60%, 50%, and 52% before RT, during RT, and end of RT, respectively. Logistic regression analysis showed that age, CCRT, and using 2% viscous lidocaine solution were independent risk factors for fungal colonization. Conclusions The results of this study demonstrated an underestimation of clinical oral candidiasis before and throughout the course of radiotherapy from using only clinical sign and symptoms. Crucial attention to detail and testing in the oral examination could improve decision making for detection of oral candidiasis in HNC patients receiving RT or CCRT.