Aim: To evaluate potential tooth colour alteration effect of five root canal sealers using a bovine tooth model and the interaction of formaldehyde with bismuth compounds. Materials and methods: A preliminary test was performed to assess the interaction of bismuth oxide in contact with formaldehyde. Seventy bovine teeth were sectioned into blocks and cavities were created. Each cavity was filled with either: AH Plus, Acroseal, MTA Fillapex, Sealer 26, Endofill and then sealed with composite. Triple antibiotic paste and unfilled samples were used as the positive and negative controls, respectively. All the specimens were immersed in water. The colour was assessed with a spectrophotometer at different intervals: immediately after filling, 24 h, 15 days and 30 days after filling. The colour change and the luminosity were calculated. The statistical analysis was performed using non-parametric Kruskal-Wallis and Dunn test (P 0.05). Results: The bismuth oxide in contact with formaldehyde exhibited a black colour. All the sealers caused tooth colour alteration after the evaluated periods, with grey staining and low luminosity values observed with Sealer 26 samples. Conclusions: All the evaluated sealers caused tooth colour alteration. The tooth staining was visually detected for Sealer 26 and might be associated with the formaldehyde released and its interaction with the radiopacifier bismuth oxide. Keywords: discolouration, formaldehyde, root canal sealers, spectrophotometer, tooth staining