Endodontic treatment focuses on debridement, disinfection, and obturation of the root canal system. Modern endodontics require knowledge of anatomy, diagnosis, treatment planning, debridement, cleaning, obturation, and coronal restoration. Modern techniques must eliminate anaerobic endodontic bacteria, which can be reduced or completely eradicated through intra-canal medication. We aimed to evaluate the knowledge and practice about using intracanal medication during endodontic treatment in Saudi Arabia and determine the preferred choice of using intracanal medication during endodontic treatment in KSA. A cross-sectional questionnaire survey carried out in KSA. Aweb-based survey was electronically distributed to endodontists, GDPs, dental interns, and undergraduate students in Saudi Arabia selected at random. The responses were gathered, and the data was then analyzed using the Chi-square test. The study included 485 participants, 221 males (45.6%) and 264 females (54.4%). 123 dental interns (25.4%), 99 dental students (20.4%), 38 endodontists (7.8%), and 225 general dental practitioners (46.4%). Most respondents (64.1%) identified disinfection as the primary function, followed by lesion healing (19.2%), reduction of pain (12.2%), coronal seal (2.7%), and dissolvent (1.9%). The indications for intra-canal medicaments are also well-documented, with a focus on non-vital tooth/pulp necrosis, persistent lesion, persistent pain, retreatment, and purulent discharge with or without sinus tract. As for participants’ knowledge scores of intracanal medication during RCT, only 3.5% of participants had high knowledge scores, 24.5% had moderate knowledge scores, and 72% had low knowledge scores. The study shows that Saudi endodontists, GDPs, dental interns, and students had inadequate knowledge about using intracanal medication during RCT.