Layered double hydroxides are members of an anionic clay family, characterised by unique two-dimensional layered structures and lend versatility in various applications. These biocompatible compounds have the potential to get intercalated with biological compounds and physico-chemically adsorbed onto organic molecules. Thus, making them important candidates for pharmaceutical and biomedical purposes. This study aims to synthesise, characterise and investigate the cellular toxicity interactions of Mg–Al LDH towards the mouse fibroblast L929 cell line. The Mg–Al LDH was synthesized by a meticulous process of co-precipitation followed by the hydrothermal method to ensure a well-defined and stable structure for suitable biological application. Characterisation techniques like Dynamic Light Scattering, Zeta potential, Scanning Electron Microscopy, Fourier transform infrared, and X-ray diffraction analysis were employed to provide deeper insights into the physiochemical properties and structural integrity of the synthesized Mg–Al LDH. The investigation of cellular interactions with the L929 fibroblast cell line served to assess the biocompatibility and potential cytotoxic effects of Mg–Al LDH. This was observed by assessing the morphological changes and evaluating the cytotoxic effects of Mg–Al LDH by utilising various techniques like phase contrast microscopy, fluorescent staining, and Giemsa staining. The cellular metabolic activity was assessed by MTT assay, and the subcellular lysosomal alteration was examined using the fluorescent staining method by the acridine orange staining. The dose-dependent response observed in the cellular interaction underscores the importance of dosage considerations for potential biomedical applications. By elucidating the dose-response relationship, this study contributes valuable information for the safe and effective usage of LDH in biomedical contexts.