Layered double hydroxide (LDH) has attracted major interest as one of the most versatile drug delivery systems especially for adsorption capacity and/or controlled delivery property of bioactive agents owing to their combining features of biohybrid. ZnAl synthesized layered double hydroxide can offer a platform to immobilize various types of bioactive compounds, particularly berberine chloride (BBC). However, the immobilization reaction of berberine chloride into ZnAl-LDH was performed by direct co-precipitation method at different ratios of BBC/LDH. BBC-ZnAl-LDH biohybrids were characterized in terms of structure, surface morphology, in vitro drug release profile and antibacterial assay against various bacterial cells. The BBC biomolecules were attached by coordinate bond. Structural and microstructural characterization confirms that interaction of BBC with ZnAl-LDH occurs by adsorption rather than intercalation of BBC within LDH layers. The BBC release profiles from BBC-ZnAl-LDH had a longer release duration compared to the physical mixture, and the drug release seemed faster with the low ratio of BBC/LDH. BBC-ZnAl-LDH can be internalized into bacterial cells. In vitro experiments in PBS medium showed that BBC-ZnAl-LDH biohybrid had higher cytotoxicity and inhibitory effects against three pathogenic bacteria; Staphylococcus aureus CIP 543154, Pseudomonas aeruginosa A22 and Bacillus subtilus ILP 1428B upon the drug release profiles and its destructive potential depends on the loading BBC on the LDH layers. Nonetheless these results prove that the prepared BBC-ZnAl-LDH biohybrids retain the anti-bacterial character of the BBC molecules and are therefore potential modified drug delivery system (DDS).