Abstract Background: Multidrug-resistant (MDR) Pseudomonas aeruginosa is a serious global health concern due to the overexpression of the efflux pump system MexAB-OprM. An innovative approach to the synthesis of nanoparticles using biogenic selenium (SeNPs) has shown promising antimicrobial properties and potential for efflux pump inhibitory activity. Objective: This study focused on the synthesis of selenium nanoparticles using leaf extract from Ziziphus spina-christi and an integrated in vitro and in silico study of their activity as efflux pump inhibitors against MDR P. aeruginosa. Methods: Clinical MDR P. aeruginosa isolates were identified using 16S rRNA gene sequencing. Green-synthesized SeNPs were characterized using UV-Vis, FTIR, and SEM. MICs were determined, and antibiotic-synergy associated FIC index (FICI) calcukations and checkerboard assauys were performed. MexAB-OprM efflux pump gene expression was quantified using RT-qPCR. Molecular docking was conducted for predicting interactions of SeNPs with MexA, MexB, and OprM proteins. Results: Biogenic synthesis SeNPs showed absorption peaks between 265 and 280 nm and had a spherical shape and a dia of 20-45 nm. SeNPs exhibited a strong antibacterial effect against multi-drug resistant (MDR) P. aeruginosa and showed a synergistic effect with imipenem (FICI: 0.25-0.375) (MIC: 16-32 (µg/mL). RT-qPCR results showed a significant downregulation of mexA, mexB, and oprM genes (2.5-4.8 fold; p<0.001). In silico molecular docking studies showed that selenium compounds have a good binding affinity to distal binding pocket of MexB with a binding energy of -7.8 to -9.1 kcal/mol and good interaction with MexA and OprM proteins. Conclusion: Biogenic SeNPs produced from Z. spina-christi significantly demonstrate the inhibition of MexAB-OprM efflux pumps in MDR P. aeruginosa, thus providing a new approach in tackling the challenge of antibiotic resistance. Given the direct antimicrobial effect and the inhibition of efflux pumps, SeNPs are likely to be useful in combination therapy for the treatment of MDR pathogens.