Abstract Bacterial biofilm-associated infections remain a major challenge in clinical settings. Innovative solutions are therefore urgently needed to improve the efficacy of antibacterial agents and to overcome biofilm tolerance. In this study, we sought to enhance the selectivity of cytotoxic small molecules via conjugation with aptamers. We developed and characterized a biofilm-specific G-quadruplex aptamer hybrid loaded with 13-(4-ethenylbenzyl)berberine iodide, which exhibited potent antibiofilm activity against Staphylococcus aureus NCTC 8325-4. The incorporation of the G-quadruplex motif into the SA31 aptamer significantly improved biofilm binding and penetration. This work presents evidence of the interaction between G-quadruplex and Staphylococcal protein A, revealing biofilm targeting properties of this motif. Mechanistic investigations revealed that treatment of the tested strain with 13-(4-ethenylbenzyl)berberine iodide was associated with transcriptional changes in pathways related to DNA replication, cell wall biosynthesis, biofilm formation and quorum sensing. Collectively, our study highlights the potential of G-quadruplex aptamer hybrids as promising delivery platform for addressing the challenges posed by biofilm-associated infections.