Biofilms are problematic in health and industry because they are resistant to various antimicrobial treatments. Ionic liquids are a novel class of low temperature liquid salts consisting of discrete anions and cations, and have attracted considerable interest as safer alternatives to organic solvents. Ionic liquids have interesting antimicrobial properties and some could find use in the development of novel antiseptics, biocides and antifouling agents. The antimicrobial and antibiofilm activity of 1-dodecyl-3-methylimiazolium iodide ([C(12)MIM]I) was studied using the clinically important bacterial pathogens, Staphylococcus aureus SAV329 and Pseudomonas aeruginosa PAO1. The ionic liquid increased cell membrane permeability in both S. aureus and P. aeruginosa cells and impaired their growth, attachment and biofilm development. The ionic liquid exhibited superior antimicrobial and antibiofilm activity against the Gram-positive S. aureus compared to the Gram-negative P. aeruginosa cells. BacLight™ staining and confocal microscope imaging confirmed that the ionic liquid treatment increased the cell membrane permeability of both the Gram-positive and Gram-negative bacteria. In addition, the antimicrobial and antibiofilm properties of [C(12)MIM]I were similar or superior to those of cetyltrimethylammonium bromide (CTAB), a well-known cationic surfactant. It is concluded that the ionic liquid induced damage to bacterial cells by disrupting cell membrane, leading to inhibition of growth and biofilm formation. Overall, the results indicate that the ionic liquid 1-dodecyl-3-methylimiazolium iodide was effective in preventing S. aureus and P. aeruginosa biofilms and could have applications in the control of bacterial biofilms.