The objective was to compare toluidine blue (TB) and autofluorescence (AF) for the detection of oral dysplasia and squamous cell carcinoma (OSCC) in clinically suspicious lesions according to conventional examination. Fifty-six clinically suspicious lesions were subjected to AF and TB examination. Data were compared using two different scenarios: in the first, mild dysplasia was considered as positive, while in the second, it was considered as negative. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), accuracy, and concordance were calculated. AF sensitivity and specificity were 70.0 and 57.7%, respectively, while TB showed a sensitivity of 80% and a specificity of 61.5%. The sensitivity increased in the second scenario in both AF (76.5%) and TB (88.2%). The specificity decreased in AF and TB, showing the same value (51.3%). PPV was higher in TB than in AF (70.6 versus 65.6%) and similarly for NPV (72.7 versus 62.5%). In the second scenario, TB PPV was 44.1% and NPV was 90.9%; AF PPV was 40.6% and NPV was 83.3%. TB showed greater accuracy than AF in the first scenario (62.5 versus 58.9%). AF and TB are both sensitive but not specific in OSCC and dysplasia diagnosis.