Objectives: Effective disinfection and antisepsis is pivotal in preventing infections within the healthcare setting. Chlorhexidine digluconate (CHG) is a widely used disinfectant/antiseptic possessing broad-spectrum antimicrobial activity; however, its penetration into bacterial biofilms and human skin is poor. The aim of this study was to investigate the antimicrobial efficacy of crude eucalyptus oil (EO) and its main component 1,8-cineole (a recognized permeation enhancer), alone and in combination with CHG, against a panel of clinically relevant microorganisms grown in planktonic and biofilm cultures. Methods: MICs and minimum bactericidal/fungicidal concentrations were determined for each microorganism grown in suspension and biofilm using microbroth dilution and ATP bioluminescence, respectively. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between CHG and EO or 1,8-cineole. Results: Antimicrobial activity was demonstrated by CHG, EO and 1,8-cineole; however, CHG was significantly more active against microorganisms in both planktonic and biofilm modes of growth (P < 0.05). Crude EO was significantly more efficacious against microorganisms grown in suspension compared with 1,8-cineole (P < 0.05). Synergistic activity was demonstrated between CHG and both EO and 1,8-cineole against suspensions of Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli and Candida albicans, and biofilm cultures of MRSA and Pseudomonas aeruginosa. Conclusions: In conclusion, CHG may be combined with either crude EO or its major component 1,8-cineole for enhanced, synergistic antimicrobial activity against a wide range of microorganisms in planktonic and biofilm modes of growth; however, the superior antimicrobial efficacy associated with crude EO alone, compared with 1,8-cineole, favours its combination with CHG.