Objectives <p>This study aimed to evaluate the antibacterial and antifungal efficacy, as well as postoperative pain levels, following non-surgical retreatment of single-rooted teeth using tricalcium silicate- or calcium hydroxide-based intracanal dressings.</p> Materials and methods <p>Forty-nine patients diagnosed with apical periodontitis were randomly assigned to two groups: tricalcium silicate (<i>n</i> = 25) and calcium hydroxide (<i>n</i> = 24). Microbiological samples were obtained before instrumentation (S1), after chemo-mechanical preparation (S2), and 10 days post-medication (S3). Total bacterial load and <i>Candida albicans</i> levels were quantified using qPCR. Postoperative pain was assessed on days 1, 3, 5, 7, and 10.</p> Results <p>No significant differences were found between the groups in demographic variables. The calcium hydroxide group showed a significantly greater reduction in total bacterial load in S3 samples. No significant differences were observed between groups regarding <i>C. albicans</i> levels or postoperative pain at any time point. However, a significant reduction in pain was noted on day 1 compared to baseline in the calcium hydroxide group.</p> Conclusions <p>Both materials were effective in reducing microbial load; however, calcium hydroxide demonstrated superior antibacterial efficacy. Antifungal activity and postoperative pain outcomes were comparable between the groups.</p> Trial registration <p>This trial was registered at Clinicaltrials.gov on 15/01/2025 (NCT06779370).</p>