A bacterial biofilm is one of the most difficult structures to eradicate and is involved in the enhancement of the virulence and resistance of bacteria. Pseudomonas aeruginosa (P.aeruginosa) is one of the most common opportunistic human pathogens that have vigorous biofilm formation, which is responsible for many life-threatening infections like those associated with cystic fibrosis, nosocomial infections, incurable burns and wounds. Several strategies have been used to fight bacterial virulence and biofilm formation through targeting the quorum sensing (QS) mechanism of communication. Curcumin is a natural component extract from turmeric and has antimicrobial and QS inhibitory (QSI) effects on many microbes and their biofilm. This study aimed to evaluate the effect of curcumin-protamine sulfate combination against the biofilm formation by P. aeruginosa(ATCC 27853). The MIC and MBC test was detected by 24 well plate method, and the MIC and MBC of Curcumin 625µg/ml, 10mg/ml. and for protamine sulfate 156µg/ml, 312µg/ml. Neither the curcumin nor the protamine sulfate exhibited antibiofilm activity when tested against the P.aeruginosa biofilm alone. However, when curcumin was combined with protamine sulfate at different concentrations, a significant reduction in biofilm formation was detected. The highest inhibition percentage was detected against 48hour biofilm when the biofilm was treated with 62.5µg/ml curcumin with 62.5µg/ml protamine sulfate using the 96-well plate method. When the biofilm was allowed to grow for 72 hours, the highest inhibition percentage, which was 43%, was measured when the biofilm was treated with 62.5µg/ml of curcumin, with 0.125mg/ml of protamine sulfate. The porcine ex-vivo model was used to confirm the previous result. The porcine skin explants was used to stimulate the biofilm growth on skin that has wounds and provides an accurate model for assessing the effects of Curcumin and protamine sulfate on mature biofilm. Explants were prepared according to the following groups: curcumin group that was treated with 100 µL of 500 µg/ml curcumin; protamine sulfate group that was treated with 100 µL of 500 µg/ml protamine sulfate; three treatment groups that were treated with 100 µL of 500 µg/ml curcumin combined with different protamine sulfate concentrations (125 µg/ml, 250 µg/ml, and 500 µg/ml); a control group that did not receive any treatment; and a vehicle group that was treated with vehicle without any treatment. The highest inhibition of biofilm was 95% when the porcine skin was treated with 500µg/ml of curcumin with 500µg/ml protamine sulfate. The obtained results from this study highly suggest that the combination of curcumin with protamine sulfate improves the effect against the biofilm, and ensures that the effect is due to the prevention of biofilm formation mainly through interruption of QS rather than killing of the bacteria. Accordingly, the tested combination can be used as an antibiofilm and anti-pathogenic agent against P.aeruginosa. Further studies are required to test the effect of this combination over the virulence factors of P. aeruginosa.