Pseudomonas aeruginosa is a remarkable opportunistic bacterium that represents a global health concern, due to its ubiquity and high levels of antibiotic resistance. Hence, the development of novel antimicrobials or alternative therapies against its infections is urgent. In this regard, anti-virulence therapies are a promising option to minimize pathogen mediated damage to the host rather than directly kill pathogenic bacteria. To date several natural and synthetic compounds had shown activity against quorum sensing regulated virulence factors of P. aeruginosa; nevertheless, the type three secretion system (T3SS), also known as injectisome, represents one of the main virulence factors of this bacterium, and a major contributor for acute infections. Importantly, the expression and activity of the injectisome appears not to be positively regulated by quorum sensing, and hence the use of specific quorum quenching enzymes does not inhibit type three secretion. In this work, we characterized the type three secretion profile of effector proteins in a collection of clinical isolates of P. aeruginosa isolated from burn patients and respiratory infections. Immunoblotting showed that the presence of an active T3SS is common in these strains, confirming it is an important determinant for its infections. Furthermore, we demonstrate that the natural compound curcumin can effectively inhibit the secretion of the main effectors ExoS and ExoU in PA01 and PA14, the main reference strains of this bacterium, as well as in representative clinical isolates. This inhibition of effectors secretion occurs despite their intracellular accumulation upon curcumin treatment, suggesting that curcumin do not work by impeding effectors expression but rather by interfering with either the assembly or the function of the T3SS.