Background: Understand the mothers about knowledge and the practices regarding their children teeth problems can support them and help them in caring among their children. Aim: To evaluate the effect of instructional guidelines on new mothers' practices regarding children's teeth problems. Subjects and method: Design: A quasi-experimental research design was used with a pre-and post-test.  Setting: The research was conducted at the antenatal outpatient clinic atSohag University Hospital, two Maternal and Child Health Centers that are attached to Sohag city and PortSaid city, which are Dar El –Salam Abed- Allah health center at Sohag city and El-Kuwait health center at PortSaid city. Subjects: A purposive sample of 260 primipara mothers was included. Two tools were used: (I) newly mothers' interviewing questionnaire (pre/post), which included (a) demographic characteristics; and (b) newly mothers' knowledge; and (II) observational checklist for oral hygiene practice (pre/post). Results: Revealed that mothers' ages ranged from 18 to 35 years, and the mean age was 23.2±4.5 years. There were marked improvements in mothers' knowledge and practices regarding children's teeth problems after instructional guidelines implementation (P=0.005). Conclusion: The instructional guidelines implementation regarding teeth problems had a positive effect on improving new mothers of preschool children's knowledge and practices. Recommendations: The instructional guidelines for new mothers should be conducted regularly to discuss and teach them different aspects about teeth problems among their children and replication of the current study with a larger sample with follow-up.