White spot formation is an undesirable complication of orthodontic fixed appliances. It is due to enamel demineralization by organic acids resulted from accumulation of cariogenic bacteria around the brackets. This lesion can jeopardize the medical and esthetic benefits of orthodontic therapy. Pro Seal (a new highly filled light-cured resin) was claimed to protect the susceptible area adjacent to bonded attachment and require no patient compliance. Therefore, the purpose of this study was to investigate the efficacy of this new sealant in preventing enamel decalcification in vivo and compare its effect with varnish and unfilled sealant using Atomic force microscopy. Thirty two premolars with brackets on their buccal surfaces were classified according to treatment with different materials into four groups (n=8 for each one, 4 maxillary and 4 mandibular); Control (non-treated), Fluoride varnish, Unfilled sealant and Filled sealant (Pro Seal). After two months the brackets were debonded and the teeth were extracted and prepared for investigation. Each sample was scanned twice at two different scan areas (50 and 10µm) at the buccal cervical third of the crown. Images were recorded with slow scan rate; the mean roughness height and total surface area were calculated for each scan area. Tapping mode images and statistical analysis showed that Pro Seal treated samples had the least damaged enamel surface of all groups and the lowest mean of roughness height and total surface area. In conclusion, Pro Seal was the most effective prophylaxis technique in preventing enamel demineralization around orthodontic brackets.