Moving away from synthetic vesicle systems, the activity of SSAs and their co-formulations was next established against biofilms of pathogenic microorganisms listed on WHO prioritised pathogens lists. These biofilms are highly complex when compared with synthetic vesicle systems. The cell membrane is comprised not only of phospholipids, but also of proteins and carbohydrates, which combined with the presence of the cell wall and extracellular matrix (ECM), adds to the chemical complexity of the cell surface. Initially, the ability of SSAs 1 -5 to inhibit biofilm formation was determined against P. aeruginosa (PAO1) biofilms through an XTT metabolic assay, used to determine the viability of cells within a biofilm (Section S4 ). As shown in Figure , 4 and 5 exhibited an enhanced antibiofilm activity over 1 and 2 at the highest SSA concentration tested (2.56 mM), with 4 also showing a greater activity over 3. At this concentration 4 and 5 were shown to completely inhibit biofilm formation. However, at lower concentrations (0.16 mM), 1 -3 showed enhanced antibiofilm activity over 4 and 5 while still maintaining biofilm inhibition > 55 %. In all cases 1 -5 exhibited an enhanced antibiofilm activity over the control TBACl. We believe that this may indicate that SSAs which contain a squaramide residue may experience an additional barrier to eliciting an antimicrobial effect that is concentration dependent. As these SSAs exhibit enhanced hydrogen bond donor properties, this may include enhanced interactions with the ECM, preventing preferential interactions of these SSAs with the bacteria. To investigate the effects of SSA co-formulation on P. aeruginosa (PAO1) antibiofilm activity, the antibiofilm efficacy of a, b and e, which incorporate those SSAs that show the greatest efficacy against this biofilm as single agents at lower concentrations (0.16 mM) were established (Figure ). In addition, a proof-of-principle experiment was also conducted with j which contains 4 and 5 to establish the potential for any enhancement in activity against a P. aeruginosa (PAO1) biofilm at lower concentrations (0.64 mM). Co-formulations e and j exhibited similar antibiofilm activities to homogenous SSA solutions, however a and b showed a loss of single agent antibiofilm activity, while higher concentrations of a were also shown to elicit a positive effect on bacterial growth.