Introduction: Bite force is a key indicator of masticatory system function and is influenced by various biological and dental factors. Alterations in pulpal innervation and periodontal sensory feedback following root canal treatment (RCT) may affect force regulation during mastication. The aim of this study was to compare pain/discomfort-limited bite force threshold values between root canal-treated and untreated teeth using a digital bite force transducer (GM10). Methods: A total of 131 patients (48 males and 83 females) participated in the study. Bite force measurements were obtained from 447 teeth (248 with RCT and 199 without RCT). Participants were instructed to bite on a digital bite fork until discomfort or pain was experienced. Therefore, the recorded values represent a pain/discomfort-limited bite force threshold rather than true maximal voluntary bite force. Peak force was recorded in Newtons (N). The device was modified with a rubber tip and a protective cover to improve safety and patient comfort. Results: Teeth without RCT demonstrated significantly higher bite force values than root canal-treated teeth (245.39 N vs. 197.06 N, adjusted p = 0.0347). Male participants exhibited significantly higher bite force values than female participants (277.62 N vs. 186.96 N, adjusted p = 0.0016). In addition, molars demonstrated significantly higher bite force values than premolars (227.83 N vs. 202.75 N, adjusted p = 0.0347). Conclusions: Root canal-treated teeth demonstrated lower bite force values than untreated teeth. However, these findings should be interpreted cautiously because of potential confounding factors.