The aim of this in vitro study was to evaluate the protective effect of short-pulsed CO2 9.3μm laser irradiation against erosion in human enamel without and combined with TiF4 and AmF/NaF/SnCl2 applications, respectively, as well as compared to the protective effect of these fluoride treatments alone. After polishing, ninety enamel samples (3 × 3mm) were used for 9 different treatment groups: 4% TiF4 gel (pH1.5, 24,533ppmF-); AmF/NaF/SnCl2 rinse (pH4.5; 500ppmF-, 800ppm Sn2); CO2 laser (average power 0.58W); CO2 laser (0.58W) + TiF4; CO2 laser (0.58W) + AmF/NaF/SnCl2; CO2 laser (0.69W); CO2 laser (0.69W) + TiF4; CO2 laser (0.69W) + AmF/NaF/SnCl2; negative control (deionized water). TiF4 gel was brushed on only once before the first erosive cycling, while samples treated with AmF/NaF/SnCl2 were daily immersed in 5ml of the solution before cycling. Laser treatment occurred with a CO2 laser (wavelength 9.3μm, pulse repetition rate 100Hz, pulse duration 14.6μs/18μs, average power 0.58W/0.69W, fluence 1.9J/cm2/2.2J/cm2, beam diameter 0.63mm, irradiation time 10s, air cooling). TiF4 was applied only once, while AmF/NaF/SnCl2 was applied once daily before the erosive challenge. Surface loss (in μm) was measured with optical profilometry immediately after treatment, and after 5 and 10days of erosive cycling (0.5% citric acid, pH2.3, 6 × 2min/day). Additionally, scanning electron microscopy investigations were performed. All application measures resulted in loss of surface height immediately after treatment. After 5days, significantly reduced surface loss was observed after applying laser irradiation (both power settings) followed by applications of TiF4 or AmF/NaF/SnCl2 solution (p < 0.05; 2-way ANOVA and Tukey test) compared to fluoride application alone. After 10days, compared to after 5days, a reduced tissue loss was observed in all groups treated with AmF/NaF/SnCl2 solution. This tissue gain occurred with the AmF/NaF/SnCl2 application alone and was significantly higher when the application was combined with the laser use (p < 0.05). Short-pulsed CO2 9.3μm laser irradiation followed by additional application of AmF/NaF/SnCl2 solution significantly reduces the progression of dental enamel erosion in vitro.