Fine-needle aspiration cytology is the standard technique to diagnose thyroid pathologies. However, this method results in a high percentage of inconclusive and false negatives. The use of time-resolved fluorescence techniques to detect biochemical composition and tissue structure alterations could help to develop a portable, minimally invasive, and nondestructive method to assist during surgical procedures. This study aimed to use fluorescence lifetimes to differentiate healthy and benign tissues from malignant thyroid tissue. The thyroid tissue was excited at 298–300 nm and the fluorescence decay registered at 340 and 450 nm. We observed fluorescence lifetimes at 340 nm emission of and for healthy tissue; and for benign lesions; and and for malignant lesions. For 450 nm emissions, we obtain lifetimes of and for healthy tissue, and for benign lesions, and for malignant lesions. Employing analysis of variance, we differentiate malignant lesions from benign and healthy tissues. In addition, we use quadratic discriminant analysis to distinguish malignant from benign and healthy tissues with an accuracy of 76.1%, sensitivity of 74.7%, and specificity of 83.3%. These results indicate that time-resolved fluorescence can assist medical evaluation of thyroid pathologies during surgeries.