This in-vitro study aimed to analyse the effect of brushing and different brushing parameters (kind of toothpaste, kind of toothbrush, brushing force) on erosive tooth wear of primary bovine enamel and dentin. Specimens were prepared from primary bovine enamel or dentin (each group n = 12) and cyclically eroded (6 × 60 s/d, citric acid, pH 2.4) and brushed with children’s toothbrushes (2 × 15 s/d) over 5 days. The brushing parameters under investigation were: toothpaste (fluoridated, fluoride-free), toothbrush (manual; rotating-oscillating and sonic, each at two different activation modes) and brushing force (1 N, 2 N). Specimens that were only eroded and not brushed served as controls. Enamel and dentin wear was quantified using widefield confocal microscopy. Statistical analysis was performed using three- and one-way ANOVAs followed by Scheffe’s (enamel) or Tamhane’s (dentin) post-hoc tests (p < 0.05). Brushing with the fluoridated toothpaste was able to significantly reduce erosive wear in enamel (by 15 to 37%, 6 of 10 groups) and in dentin (by 58 to 72%, all groups), while brushing with the fluoride-free toothpaste was not different from the controls. Considering the kind of toothpaste and brushing force, slight differences between the toothbrushes were observed in enamel, but not in dentin. Within the same toothbrush and activation mode, almost no differences between 1 and 2 N brushing force were detected. In conclusion, erosive tooth wear on primary bovine dental hard tissue mainly depends on the kind of toothpaste, rather than on the kind of toothbrush and the brushing force.