This study investigates the bactericidal efficacy and penetration ability inside dentinal tubules of calcium hydroxide (CH) and a modified tri-antibiotic paste (ciprofloxacin, metronidazole, clarithromycin) (TAP) during regenerative endodontic procedures (REPs). The blood clot serving as a biological scaffold was introduced into the root canal to assess its influence on bacterial regrowth. Forty-four human extracted teeth were infected with E. faecalis and divided in four experimental groups (N = 10) with positive and negative controls (N = 4). Samples were treated with either CH or TAP as intracanal dressing. Bacterial viability and depth of penetration were evaluated using confocal laser scanning microscopy (CLSM) after fluorescent vital staining. The same analysis was performed with or without blood clot exposure and the data were analyzed by one-way ANOVA and a post hoc Bonferroni test (p < 0.05). TAP demonstrated significantly stronger bactericidal activity than CH (p = 0.008). However, its efficacy significantly decreased in the presence of blood clot (p = 0.032). CH showed a moderate antibacterial effect, with its efficacy reduced in the presence of blood. Blood clot exposure consistently reduced the bactericidal efficacy in both groups (p = 0.01) and the dye penetration in CH group (p = 0.041). In conclusion, TAP demonstrated superior antibacterial performance compared to CH and blood clot exposure seemed to decrease antimicrobial efficacy and depth of disinfection during REPs.