This study aims to investigate whether toothbrushes with fluoride-infused bristles have any (re)mineralisation effects on bovine enamel. Bovine incisors (N = 160) were extracted, and the buccal side of the crown was cut into dimensions of ~5 mm × 5 mm with a low-speed saw. These specimens were randomly allocated into four groups: half (80 teeth) were stored in demineralising solution (DM), and the other half were stored in deionised water (DW) for 96 h. Then, they were brushed with a force of 2.0 ± 0.1 N for five min with a manual toothbrush with either fluoride-infused (TF) or regular (TR) bristles. Microhardness (Vickers), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), and scanning electron microscopy (SEM) were used to investigate the surfaces of the bovine enamel specimens before and after brushing. Two-way ANOVA was used to analyse the hardness data, and the pairwise comparison method was used to analyse the Ca/P ratio, for each group at α = 0.05. The results show that brushing with either of these toothbrushes increased the Vickers microhardness on DM and DW enamel (p < 0.001), whereas hydroxyapatite was revealed in all groups by XRD. The DM samples showed a significant increase (p < 0.05) in the Ca/P ratios after brushing with TR and TF. Conversely, under DW conditions, these ratios decreased significantly after brushing. In terms of the F atomic%, TF increased significantly. SEM revealed mineral deposition in the DM groups after toothbrushing. To conclude, toothbrushing effectively induces the microhardness of sound and demineralised enamel, while fluoride-infused bristles might be able to retain fluoride on the enamel surface.