Complex stalled wounds feature an alkaline milieu that favors tissue destruction and microbial growth. The presence of bacteria in turn perpetuates the inflammatory response. However, only limited knowledge exists of pH dependency on the antibacterial efficacy of polyhexamethylene biguanide (PHMB) or the influence of surfactants or delivery vehicle used in antiseptic formulations. So far, PHMB alone has been shown to protect the keratinocytes from bacterial damage in such a co-culture system as well as exhibiting increased antimicrobial activity at higher pH values. Here, the interaction of PHMB with the surfactants macrogolum and undecylenamidopropyl betaine that are most commonly used as additives in antiseptics and rinsing solutions such as Lavasept and Prontosan has been explored in addition to the PHMB-containing biocellulose dressing Suprasorb X + PHMB. Undecylenamidopropyl betaine was found to lower the antimicrobial activity of polihexanide in the co-culture system, while macrogolum and the biocellulose increased polihexanide efficiency to reduce Staphylococcus aureus especially in the presence of serum. The increasing antibacterial efficacy of PHMB with rising pH was not altered by undecylenamidopropyl betaine, macrogolum, or the biocellulose. The results suggest that application of PHMB with macrogolum or by delivery through a biocellulose dressing might be advantageous for management of wound infections.