The possibility of having a commercial product such as waxed floss with an additional agent for controlling and preventing caries is promising. The aim of this research was to determine the uptake of fluoride on tooth enamel in situ after the utilization of a dental floss with fluoride incorporated into the wax. One hundred blocks of bovine enamel were artificially demineralized and randomly separated into a Control Group (C) and a Test Group (T). The dental blocks in group T were mounted two-by-two simulating proximal contacts and were fixed into intra-oral lower arch devices. Eight volunteers with a similar salivary flow and buffer capacity wore devices with the enamel blocks for eight days. During this period of time the subjects applied a 25 cm long portion of a mint waxed floss with fluoride (0.15 mgF/m, Oral-B) between the blocks, 3 times a day for 2 minutes after each meal. At the same time, brushing was carried out with a fluoride-free toothpaste. The alkali-soluble fluoride (CaF2) formed on the enamel was extracted using the Caslavska et al. method and measured with the Orion 96-09 electrode-specific and the EA 720 ion analyzer. The results showed that the group that had the most CaF2 on the enamel (median, minimum and maximum in microF/cm2) was Group T (3.00, 2.11 and 4.00), which differed significantly (p < 0.01) from group C (0.26, 0.10 and 0.69). It was concluded that fluoride uptake on enamel was 11.54 times higher after use of dental floss with fluoride in this study.