This study aimed at evaluating the efficacy of a novel silver-citrate root canal irrigation solution (BioAKT) on smear layer removal, sealer penetration after root canal instrumentation and antibacterial activity. Single-root teeth were endodontically treated, sealed with an epoxi-amine resin sealer and irrigated using: Group I: 5.25% sodium hypochlorite (NaOCl); Group II: silver-citrate solution (BioAKT); Group III: phosphate buffer solution (PBS); Group IV: 17% ethylenediaminetetraacetic acid (EDTA). Smear layer removal and silver deposition at the coronal, middle and apical portion of each canal were analyzed using scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDS). Sealer penetration into dentinal tubules at coronal, middle and apical portion was assessed through dye-assisted confocal microscopy (CSM). Both SEM and CSM micrographs were evaluated by two examiners (κ = 0.86), who were blind to the irrigation regimens; scores were given according to the degree of penetration of the sealer. Data analysis included Pearson’s x2 and Sidak’s multiple comparisons. Dentin discs were polished and sterilized. Enterococcus faecalis biofilms were grown using a continuous-flow bioreactor under anaerobic conditions for 72 h. Specimens were irrigated with the tested solutions, and bacterial viability was assessed using a tetrazolium salt assay (MTT). Statistical analysis included one-way ANOVA and Student’s post-hoc t-test (p < 0.05). BioAKT and EDTA were the most efficient solutions both in removing the smear layer and allowing sealer penetration. However, at the apical portion BioAKT performed significantly better compared to EDTA both in smear layer removal and sealer penetration (p < 0.05). BioAKT and NaOCl showed comparable antibacterial effect (p = 0.53). In conclusion, BioAKT represents a suitable smear layer removal agent, which allows for reliable sealer penetration at the apical portion of the root canal system and offers significant antibacterial properties.