The aim of this study was to evaluate color change, mineral composition and topography of the enamel after the pre-treatment with non-thermal atmospheric plasma (NTAP) followed by 38% hydrogen peroxide (HP) bleaching. Buccal enamel of bovine incisors teeth was stained with black tea solution and then divided into five groups (n = 12), according to the plasma application time: PL1 (one minute) or PL2 (2 min) and number of HP applications: HP1 (one) and HP3 (three). The following groups were investigated: HP3, PL1+HP1, PL1+HP3, PL2+HP1 and PL2+HP3. Four color measurements: initial (untreated), after staining and after bleaching (first and second sessions), were performed using an intraoral spectrophotometer (Vita Easyshade). Energy Dispersive X-ray Spectroscopy (EDX) was used to analyze the mineral composition of the enamel before and after two bleaching sessions. Laser confocal microscopy was used to analyze the enamel topography. The pre-treatment with NTAP did not improve the whitening results. The use of NTAP preserves the concentration of calcium and phosphorus, only HP3 group experimented a significant decrease in the mineral concentration. All the bleached groups, with or without NTAP treatment increased their enamel roughness. The application of NTAP did not influence the color change, but it was important to maintain the mineral composition of bleached teeth. All the groups increased their roughness level.