Objective: To compare the deciduous enamel fluoride concentration between two tooth cleaning methods prior to fluoride varnish application. Methods: A clinical trial was carried out in 20 preschool children (age 4-6 years, 10 boys and 10 girls) with the consent from their parents. Two different tooth cleaning methods, i.e. toothbrushing without toothpaste and tooth cleaning with gauze, were randomly assigned to each child using a half mouth design. After that, 2.26% fluoride varnish (Duraplat®) was applied to the teeth of all children by the same operator. After 24 hour fluoride varnish application, enamel biopsy by acid-etched technique was performed on the intact baccal surface of teeth 74 and 84, respectively. Etch enamel sample was analysed for the F- and Ca2+ by using fluoride electrode and atomic absorption spectrophotometer. Results: The deciduous enamel fluoride concentration which cleaned by toothbrush and gauze were 791±104ppm and 737±94ppm, respectively, which was not statistically different (p>0.05, t-test). Conclusions: Tooth cleaning prior to fluoride varnish application, either with toothbrushing or gauze induces similar deciduous enamel fluoride concentration.