In this study, we sought to evaluate the influence of cigarette smoke and pH cycling on the chemical composition and surface/cross-sectional enamel microhardness. A total of 40 dental blocks obtained from bovine incisors were divided into four groups (n=10): no treatment (control); exposure to cigarette smoke (CS); exposure to pH cycling (PC); and exposure to cigarette smoke and pH cycling (CS-PC). The samples were analyzed by synchrotron radiation micro X-ray fluorescence, bench mode X-ray fluorescence, as well as surface microhardness (SMH) and cross-sectional microhardness (CSMH) testing. The SMH results were submitted to analysis of variance (ANOVA) and Tukey’s test. The CSMH results were evaluated using split-plot ANOVA and Tukey’s test. A high amount of Cd and Pb and traces of Ni and As were observed in enamel and dentin after exposure to cigarette smoke (CS and CS-PC). The SMH and CSMH of CS were statistically higher when compared with the control. The PC and CS-PC showed lower SMH and CSMH. We conclude that exposure to cigarette smoke promoted heavy metal deposition in enamel/dentin. In addition, it increased the enamel microhardness but did not promote a protective effect on the in vitro development of caries. The clinical significance of this work is that there is significant bioaccumulation of heavy metals from cigarette smoke on the surface and in the enamel and dentin.