Bacterial biofilms contribute to increased pathogenesis and bacterial resistance. Biofilms can enhance pathogenicity by shielding bacteria from the immune system and antibiotics, and they are associated with persistent infections. Additionally, the antibiotic resistance mechanisms within biofilms make them challenging to treat, emphasizing the need for strategies to be addressed. Mitigating bacterial virulence is a promising strategy that could ease their eradication by host immunity without stressing bacteria to induce resistance. The merits of this strategy are augmented when using safe anti-virulence candidates in proper formulations. The current study aimed to evaluate the antibiofilm and anti-virulence efficacy of curcumin–zein nanoparticles against Pseudomonas aeruginosa. In vitro investigations were performed to assess the effect of nanoparticles on biofilm formation, bacterial motility, and production of virulence factors, including proteases, hemolysins, and pyocyanin, in comparison to bulk curcumin. Furthermore, the effect on the expression of the genes that encode quorum sensing (QS) systems that regulate bacterial virulence was assessed. An in silico study was done to evaluate the affinity of curcumin to QS receptors. Additionally, an in vivo protection assay was performed to evaluate the inhibitory effect of our preparation on diminishing the P. aeruginosa’s capacity to induce pathogenesis. The results showed significant antibiofilm and anti-virulence activities of the curcumin–zein nanoparticles compared to bulk curcumin. These anti-virulence activities were attributed to the curcumin’s interfering with the P. aeruginosa QS systems that regulate its virulence. In conclusion, curcumin acquires significant anti-QS, anti-virulence, and antibiofilm activities that are vastly enhanced upon loading on zein nanoparticles.