Abstract: Pseudomonas aeruginosa is an important multidrug-resistant human pathogen by dint of its high intrinsic, acquired, and adaptive resistance mechanisms, causing great concern for immune-compromised individuals and public health. Additionally, P. aeruginosa resilience lies in the production of a myriad of virulence factors, which are known to be tightly regulated by the quorum sensing (QS) system. Anti-virulence therapy has been adopted as an innovative alternative approach to circumvent bacterial antibiotic resistance. Since plants are known repositories of natural phytochemicals, herein, we explored the anti-virulence potential of Azorella atacamensis, a medicinal plant from the Taira Atacama community (Calama, Chile), against P. aeruginosa. Interestingly, A. atacamensis extract (AaE) conferred a significant protection for human lung cells and Caenorhabditis elegans nematodes towards P. aeruginosa pathogenicity. The production of key virulence factors was decreased upon AaE exposure without affecting P. aeruginosa growth. In addition, AaE was able to decrease QS-molecules production. Furthermore, metabolite profiling of AaE and its derived fractions achieved by combination of a molecular network and in silico annotation allowed the putative identification of fourteen diterpenoids bearing a mulinane-like skeleton. Remarkably, this unique interesting group of diterpenoids seems to be responsible for the interference with virulence factors as well as on the perturbation of membrane homeostasis of P. aeruginosa. Hence, there was a significant increase in membrane stiffness, which appears to be modulated by the cell wall stress response ECFσ SigX, an extracytoplasmic function sigma factor involved in membrane homeostasis as well as P. aeruginosa virulence.