Pseudomonas aeruginosa is a non-fermentative, gram-negative bacterium that is one of the most common pathogens responsible for hospital-acquired infections worldwide. The management of the infections caused by P. aeruginosa represents a huge challenge in the healthcare settings due to the increased emergence of resistant isolates, some of them resistant to all the currently available antimicrobials, which results in elevated morbimortality rates. Consequently, the development of new therapeutic strategies against multidrug-resistant P. aeruginosa is urgent and needful. P. aeruginosa is wellrecognized for its extreme genetic versatility and its ability to produce a lush variety of virulence factors. In this context, pseudolysin (or elastase B) outstands as a pivotal virulence attribute during the infectious process, playing multifunctional roles in different aspects of the pathogen-host interaction. This protein is a 33-kDa neutral zinc-dependent metallopeptidase that is the most abundant peptidase found in pseudomonal secretions, which contributes to the invasiveness of P. aeruginosa due to its ability to cleave several extracellular matrix proteins and to disrupt the basolateral intercellular junctions present in the host tissues. Moreover, pseudolysin makes P. aeruginosa able to overcome host defenses by the hydrolysis of many immunologically relevant molecules, including antibodies and complement components. The attenuation of this striking peptidase therefore emerges as an alternative and promising antivirulence strategy to combat antibiotic-refractory infections caused by P. aeruginosa. The anti-virulence approach aims to disarm the P. aeruginosa infective arsenal by inhibiting the expression/activity of bacterial virulence factors in order to reduce the invasiveness of P. aeruginosa, avoiding the emergence of resistance since the proliferation is not affected. This review summarizes the most relevant features of pseudolysin and highlights this enzyme as a promising target for the development of new anti-virulence compounds.