Abstract Background Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed. Objectives To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections. Methods We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide. Results The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive. Conclusions The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.