Biofilms formed by several bacterial strains still pose a significant challenge to healthcare due to their resistance to conventional treatment approaches, including antibiotics. This study explores the potential of loading natural extracts with antimicrobial activities into β-cyclodextrin (βCD) nanoparticles, which are FDA-approved and have superior biocompatibility owing to their cyclic sugar structures, for biofilm eradication. An inclusion complex of βCD carrying essential oils (BOS) was prepared and characterized with regard to its physicochemical properties, antimicrobial efficacy, and antibiofilm activities. Encapsulation of BOS into βCD significantly enhanced the antimicrobial activity of BOS by 4-fold against Gram-positive ( and ) and by 8-fold against Gram-negative ( and ) bacteria, with minimum inhibitory concentrations ranging from 2.5 to 5 mg mL. Furthermore, the BOS-βCD complex demonstrated a dual-action against bacterial biofilms where it prevented biofilm formation and disrupted established biofilms. This resulted in a significant reduction in biofilm biomass, with prevention and disruption rates reaching up to 93.78% and 82.17%, respectively. Additionally, the formula revealed an excellent biocompatibility profile with no induction of oxidative stress in human skin fibroblast cells. Our findings suggest that βCD nanoparticles loaded with BOS essential oils hold promise as an effective formula for preventing the formation of bacterial biofilms and combating preformed ones for use in relevant medical applications.