This study aimed to analyze the presence of defects within the adhesive interface formed with five bioactive dental materials and caries-affected dentin concerning the timing of radiotherapy (before or after the restorative procedures) by micro-CT. A total of 96 carious human molars were randomly allocated into the following groups based on the timing of the radiotherapy sequence: radiotherapy followed by restoration (RT1) or restoration followed by radiotherapy (RT2). Then, six subgroups were established within these groups based on the type of materials used (n=8). Following cavity preparation and caries removal, a universal adhesive (G-Premio Bond) was administered in self-etch mode or accompanied by applying suitable cavity conditioners according to the manufacturers' guidelines. Subsequently, restorations were performed using five bioactive restorative materials (resin-modified glass-ionomer [Fuji II LC], high-viscosity glass-ionomer hybrid [EQUIA Forte HT], giomer [Beautifil II], alkasite [Cention N], and dual-cure bulk-fill composite [Activa Bioactive Restorative]) and a conventional microhybrid resin composite (Filtek Z250). The radiotherapy regimen encompassed 60 Grays (Gy) administered at a rate of 2 Gy/day over 6 weeks, 5 days a week. Micro-CT analysis was employed to assess adhesive defects at the interface between caries-affected dentin and the restorations. The data were analyzed using Kruskal-Wallis, Mann-Whitney U, and Dunn tests (α=0.05). RT2 caused significantly higher adhesive defects than RT1 for the Filtek Z250 and Activa Bioactive Restorative subgroups (p<0.05). For RT1, no significant differences were found in adhesive defects among all tested subgroups (p>0.05). By contrast, for RT2, adhesive defects were significantly higher for the Activa Bioactive Restorative and Cention N subgroups than for the EQUIA Forte HT and Beautifil II subgroups (p<0.05). When using most bioactive restorative materials, the timing of radiotherapy had no significant influence on the adhesive interface. Regarding restoration following a radiotherapy protocol, a favorable impact was identified with high-viscosity glass ionomer hybrid cement and giomer bioactive restorations compared with dual-cure bioactive bulk-fill composite and alkasite restorations.