Objectives: The purpose of this investigation was to evaluate the substantivity of chlorhexidine (CHX) within the root canal system and to assess how long the CHX remains antimicrobially effective. Materials and Methods: Sixty freshly extracted intact human lower premolars with single root were collected, stored, and disinfected. The roots were divided randomly into two treatment groups: experimental (n = 30) and control (n = 30). Experimental samples (n = 30) were treated with 2% CHX and control samples (n = 30) were treated with sterile saline for 10 min. The samples were obturated with AH 26 sealer and gutta percha using lateral condensation technique. Each group was further randomly divided into three separate subgroups and stored in sterile saline for 1 week, 3 weeks, and 6 weeks. After the respective storage periods, all specimens were halved, and canal dentin was ground out with a peeso reamer. To determine whether the CHX from the dentin samples remained antimicrobial, the extracts from the experimental and control groups were mixed with the culture of Enterococcus faecalis. Results: Results were analyzed using one-way analysis of variance and Duncan's multiple range test, which showed statistically significant difference between the control and experimental groups with ultraviolet spectrophotometer for all storage periods but only till 3 weeks with antimicrobial tests. Conclusion: It can be concluded from this study that CHX is retained in the root canal in antimicrobial effective amounts for up to 3 weeks.