An antibiotic ampicillin was introduced in InP/ZnSexS1–x/ZnS-based quantum dot light-emitting diodes (QLEDs) to achieve charge balance via band alignment. The charge distribution on ampicillin facilitates the generation of an interfacial dipole, and the polarity of ampicillin is controlled by varying the pH of the charge transport layers of the QLEDs. In ZnO electron transport layers (ETLs), ampicillin is anionic at pH ∼7.5, and the weak interfacial dipole reduces the conductivity of the ETL. The anionic ampicillin in ZnO ETL also increases the energy of the lowest unoccupied molecular orbital of ZnO from 3.55 to 3.80 eV. In PEDOT:PSS hole injection layers (HILs), ampicillin is cationic at pH ∼4, and a strong interfacial dipole increases the conductivity of the HIL. The cationic ampicillin in PEDOT:PSS HIL also decreases the energy of the highest occupied molecular orbital of PEDOT:PSS from 5.25 to 5.30 eV. The improved charge balance achieved through energy band alignment enhanced the external quantum efficiency from 0.9% to 4.7%. This is the first report of a QLED in which same antibiotic material was applied to both the ETLs and HILs to enhance the band alignment, which resulted in charge balance.