The aim of this study was to investigate the effectiveness of both waiting periods between acid exposure and tooth brushing and fluoride applications in preventing toothbrush abrasion of acid-softened enamel surfaces. The study, on 5 subjects, had an in situ crossover design with experimental periods of 5 days each. Human enamel samples were recessed in mouth appliances and at the end of each experimental period, enamel loss was determined profilometrically. Specimens were eroded extraorally (2 × 20 min per day; 0.05 M citric acid), standardized brushing (2 × 30 s per sample per day; powered toothbrush) was performed in situ. The groups were: (1) erosion only, (2) brushing with fluoride-free toothpaste directly after, (3) 2 h after, or (4) before erosion; fluoride application was either (5) brushing with a fluoride toothpaste or (6) brushing with a fluoride toothpaste or gel, and rinsing with a fluoride mouth rinse. Enamel loss was (1) 45.2 ± 10.8, (2) 79.3 ± 7.8, (3) 81.7 ± 9.5, (4) 69.7 ± 13.8, (5) 51.5 ± 13.0, and (6) 41.2 ± 1.8 µm. Brushing without fluoride increased the enamel loss significantly (p ≤ 0.001), waiting for 2 h had no protective effect, and brushing before erosion decreased enamel loss values only by 12% (n.s.). In the fluoride groups, enamel loss was significantly lower than after brushing with the fluoride-free toothpaste and comparable to values after erosion only (n.s.). Waiting periods had only a minor effect, whilst the application of fluoride appeared promising.