To evaluate the bleaching ability, the effect on enamel surface and cytotoxicity of novel tooth-whitening formulations containing papain, ficin, or bromelain. Forty bovine dental discs (6 cm ×4 cm) were pigmentated and randomly allocated into the following groups (n = 10): Group 1, 20 wt% carbamide peroxide (control); group 2, 1% papain-based whitening; group 3, 1% ficin-based whitening; and group 4, 1% bromelain-based whitening. The whitening gels were prepared and applied on the enamel three times per day once a week, for 4 weeks. Color measurement was obtained by CIEDE2000. Enamel Knoop microhardness and roughness were evaluated. The WST-1 assay was used to evaluate the cell viability of mouse fibroblast cells (L929). Data were statistically analyzed by one-way analysis of variance (ANOVA) and Student Newman Keuls's post hoc test at α = 0.05 significance level. Bromelain, ficin-based, and carbamide peroxide bleaching gels showed a similar color change (p < 0.001). Higher enamel hardness decrease and higher enamel roughness were caused by the carbamide peroxide (p < 0.05). The experimental whitening gels did not affect cell viability. Tooth bleaching gels containing bromelain, papain, or ficin have substantial clinical potential to be used in the development of peroxide-free tooth whitening gels.