The increasing prevalence of antimicrobial resistance has created a need for the development of innovative antimicrobial strategies beyond traditional antibiotics. Layered double hydroxides, with their tunable chemical composition and controlled ion release capabilities, have emerged as promising candidates for facing multidrug-resistant pathogens. In this study, layered double hydroxides were synthesized using co-precipitation and double microemulsion methods to produce nanoparticles with distinct particle sizes (diameter and thickness) and intercalated anions. Their antimicrobial activity was evaluated against different bacterial and fungal strains, including Staphylococcus aureus and Candida albicans . The results revealed that layered double hydroxides with smaller particle sizes and intercalated bromide anions demonstrated superior antibacterial efficacy, attributable to enhanced ion release and increased interaction with microbial membranes. Notably, layered double hydroxides prepared by double microemulsion and containing Mg(II), Cu(II), Al(III) and bromide anions exhibited the highest antimicrobial activity, highlighting the impact of particle dimensions and intercalated anion properties on performance. This work highlights the potential of layered double hydroxides based materials as versatile antimicrobial agents, offering a sustainable solution to address the challenges of antimicrobial resistance in clinical and environmental applications. • Antimicrobial LDHs with tunable anions and particle sizes were synthesized. • m-MgCuAl-Br exhibited higher antimicrobial efficacy against bacteria and yeasts. • Smaller particles and guest anions improve ion release and microbial interaction. • Synthesis and intercalated anions affect LDH stability and antimicrobial performance. • LDHs represent a sustainable alternative to conventional antimicrobials.