Pseudomonas aeruginosa is an opportunistic bacillus possessing numerous virulence factors. It is a common pathogen in burn wounds and eye and lung infections and is highly resistant to antibiotics, causing serious health threats. Resistance is likely related to its ability to form biofilms; therefore, eradication of biofilm formers is a challenging and high-priority area. In this study, we have tested the antibiofilm activity of synthetic tryptophan-rich, cationic peptides, PuroA (FPVTWRWWKWWKG-NH2), P1 (RKRWWRWWKWWKR-NH2), and W7 (WRWWKWW-NH2), whose design is based on the tryptophan-rich domain of puroindolines (PIN). PuroA, P1 and W7 were tested for their ability to inhibit the initial cell attachment to a surface and/or impact other factors in biofilm formation. At MIC levels, P1 and W7 showed around 32% and 53% reduction, in initial cell attachment of P. aeruginosa ATCC 9721. The peptides were also effective in reducing metabolically active biofilm biomass by around 56% and 72% at MIC and reduced the cell surface hydrophobicity by about 40% and 50%, respectively. Production of rhamnolipid, a significant factor in biofilm formation especially in the detachment stage, was also reduced by around 50%, 55% and 58% by PuroA, P1 and W7, respectively, at their MICs. All peptides impacted the production of pyocyanin, a virulence factor as well as an important factor in biofilm formation. Pyocyanin production by P. aeruginosa ATCC 10145 (a pyocyanin producer) was inhibited by around 49%, 55%, and 58% at MIC levels of PuroA, P1, and W7, respectively. P. aeruginosa ATCC 10145 is a motile bacterium and its motility (swimming, swarming, and twitching) could be hindered by two peptides, P1 and W7, causing reductions in swarm zone by approximately 13% and 22%, swim zone by about 70% and 64% and twitching zone by about 28% and 17%, respectively, at their MICs. The results establish the potential of these TRP-rich cationic peptides in combating persistent infections caused by P. aeruginosa.