Background: Suppression of quorum sensing (QS) that regulates many virulence factors, including antimicrobial resistance, in bacteria may subject the pathogenic microbes to the harmful consequences of the antibiotics, increasing their susceptibility to such drugs. Aim: The current study aimed to make an aqueous crude extract from the soil Proteus mirabilis isolate with the use of the gas chromatography-mass spectrometry (GC-MS) technique for its analysis, and then, study the impact of the extract on clinical isolates of Pseudomonas aeruginosa. Methods: Preparation of crude extracts from P. mirabilis (both organic and aqueous), which were then analyzed by GC-MS to detect the bioactive ingredients. Furthermore, the extract’s capability to interfere with both the expression of the QS of P. aeruginosa and its antibacterial resistance was examined. Results: The highest GC-MS peak (37.11%.) appeared for 1,3-benzodioxole, 4-methoxy-6-(2-propenyl), along with the presence of other components of antibacterial activities. When the aqueous extract was added to the culture of two multi-drug resistant (MDR) P. aeruginosa, a significant reduction in the expression of the QS regulatory gene LasI occurred, indicating its interference with QS. Moreover, upon adding the extract to the culture of P. aeruginosa (MDR) and then subjecting it to Amikacin and Colistin, already not effective on the bacteria, the isolates became more susceptible to these antibiotics showing zones of inhibition of 25 and 17 mm, respectively. Conclusion: The crude aqueous extract of the soil P. mirabilis isolate might be a potential producer of QS inhibitors with antibacterial activities that render the MDR P. aeruginosa more susceptible to antibiotics to whom they already exerted resistance.