Introduction: This study aimed to evaluate the effect of oral hygiene instruction programs on the knowledge, attitude, performance, and DMFT index in pregnant women. Materials & Methods: In this clinical trial, 120 pregnant women in Fereydoun Shahr (Isfahan Province) were selected by cluster sampling method. The subjects were randomly divided into intervention and control groups. Educational intervention was conducted through four 2-hour sessions in the field of oral health in four groups with 11 subjects and two groups with eight subjects in the intervention group. The data of both groups were collected before, four weeks and three months after the intervention. The data were analyzed with SPSS 21, using independent-samples t-test, chi-squared test, and repeated measurements (α = 0. 05). Results: Of 120 subjects, 60 were in the control group, with the remaining 60 in the intervention group. After the educational intervention, after three months, the means of knowledge scores in the control and intervention groups were 8. 02 ± 1. 70 and 14. 15 ± 1. 05, respectively. The mean attitude scores were 30. 45 ± 3. 76 and 36. 00 ± 2. 33, and the mean performance scores were 35. 17 ± 3. 53 and 41. 43 ± 2. 40 in the control and intervention groups, respectively. Furthermore, the means of PI were 3. 28 ± 0. 81 and 2. 95 ± 0. 75 in the control and intervention groups, respectively. A repeated measure design was used to study the effect of time, group, interaction between the groups, and time on knowledge, attitude, performance, and DMFT index. The results showed a significant increase in the knowledge, attitude and performance of mothers in the intervention group compared to the control group. Evaluation of the DMFT index showed that time and group interaction was significant concerning decayed and filled teeth. Conclusion: The results showed that the educational program had a significant effect on improving the knowledge, attitude, and oral health parameters of pregnant women.