Antibiotic-resistant bacterial infections are a global health problem. A commonly-used antibiotic Penicillin G was incorporated into ZnAl-layered double hydroxides (PNG/LDH) with a varied amount of PNG. PNG/LDH nanocomposites were well characterized in structure and composition using elemental analysis, X-ray diffraction pattern, Fourier transform infrared spectroscopy and TEM images, revealing that PNG were mostly adsorbed on the LDH surfaces at a lower PNG loading but some were intercalated into LDH interlayers at a higher PNG loading. The typical release profile of PNG and Zn from PNG/LDH was a quick release, followed by a sustainable slow release. The antibacterial tests against Escherichia coli demonstrated that PNG/LDH with a suitable composition synergistically improved bacterial inhibition compared with free PNG and pristine LDHs. In specific, PNG/LDH with much higher cost-effectiveness showed a potent antimicrobial activity and maintained the activity for up to 10 days, significantly elongating the antibacterial effect compared with just 1 day for free PNG in the same conditions. Our results suggest suitable composition of nanoparticle carriers and antibiotics could significantly enhance antibacterial activity of antibiotics for a long period via the synergistic effect between carrier and antibiotics, a potential approach to overcome the bacterial resistance to antibiotics.