<i>Background:</i> The formation of biofilms is crucial in the pathogenesis of many acute and subacute microbial infections, including chronic wounds and foreign-body-related infections. Topical antimicrobial therapy with chemical antiseptics or physical treatment with tissue-tolerable plasma (TTP) may be promising to control bacterial infection. <i>Methods:</i> We assessed the efficacy of 0.1% chlorhexidine digluconate (CHX), 0.02 and 0.04% polihexanide (polyhexamethylene biguanide, PHMB) and of TTP against <i>Pseudomonas aeruginosa</i> SG81 biofilm grown in microtitre plates (polystyrene) and on silicone materials in an artificial wound fluid. <i>Results:</i> Overall, PHMB was as effective as CHX in reducing the total amount of biofilm (gentian violet assay) and in reducing the bacterial metabolism in biofilms (XTT assay). TTP also led to a significant reduction in colony-forming units. <i>Conclusion:</i> The antimicrobial activity of PHMB in biofilms is comparable to that of CHX. TTP could become an interesting physical alternative to chemical antisepsis in the future.