Aim The aim of the study was to investigate the influence of cavity cleaning and conditioning on marginal integrity of directly placed post-endodontic composite class-I-restorations in vitro. Methodology A total of 168 fully intact teeth without caries or fillings received pre-endodontic composite restorations (class-II) after their extraction. Occlusal endodontic access-cavities were prepared, and root canals were instrumented and filled with gutta-percha and an epoxy resin-based sealer. Prior to post-endodontic class-I-restoration, access cavities were completely contaminated with sealer, cleaned with alcohol and pre-treated as follows: cleaner only (alcohol), glycine-polishing, Al2O3 sandblasting, carbide bur (immediate as well as delayed restoration). A positive control (not contaminated with sealer and adhesive used) and negative control (cleaner used but no adhesive) were established. Half of the teeth from each group were subjected to thermocycling and mechanical loading (TCML). Marginal integrity of post-endodontic restoration was evaluated in oro-vestibular or mesio-distal sections after AgNO3 dye penetration (DP) by standardized photomacroscopic imaging and expressed in per cent of margin length along all segments and separately for enamel, dentine and composite, respectively. Results were analysed non-parametrically (alpha = .05). Results No restorations or teeth fractured or debonded completely. Without TCML, the median DP of all segments was significantly higher for the negative control compared with all other groups in oro-vestibular cutting direction (53%; p = .002) and in mesio-distal cutting direction (51%; p <= .041). The other groups without TCML revealed 16%-24% DP (oro-vestibular) and 12%-24% DP (mesio-distal). With TCML, the median DP in oro-vestibular cutting direction for all segments ranged between 48% and 62% for all groups, a significant difference was only observed between glycine-polishing and carbide bur (p = .041). In mesio-distal cutting direction, the median DP in negative control was 69% with TCML and significantly higher compared with all other groups (p = .002). For all other groups, the median DP of all segments ranged between 28% and 40% with TCML without significant differences. Error rates method (k = 7) revealed a significant influence of TCML in general on penetration of all segments in both oro-vestibular and mesio-distal cutting directions. Conclusion Additional access cavity pre-treatment after alcohol cleaning did not improve the marginal integrity of post-endodontic composite restorations. Thorough cleaning of the access cavity with alcohol seems to assure an acceptable marginal integrity to the tooth and restorative composite. Copyright (c) 2022 International Endodontic Journal. Published by John Wiley & Sons Ltd.