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Abstract Background Multidrug-resistant organisms (MDROs) such as vancomycin-resistant enterococci (VRE) and carbapenemase-producing Enterobacteriaceae (CPE) are associated with prolonged hospitalisation, increased medical costs, and severe infections. Faecal microbiota transplantation (FMT) has emerged as an important strategy for decolonisation. This study aimed to evaluate the genetic response of MDROs to FMT. Methods A single-centre prospective study was conducted on patients infected with VRE, CPE, or VRE/CPE who underwent FMT between May 2018 and April 2019. Genetic response was assessed as the change in the expression of the resistance genes VanA, bla KPC, bla NDM, and bla OXA on days 1, 7, 14, and 28 by real-time reverse-transcription polymerase chain reaction. Results Twenty-nine patients received FMT, of which 26 (59.3%) were infected with VRE, 5 (11.1%) with CPE, and 8 (29.6%) with VRE/CPE. The mean duration of MDRO carriage before FMT was 71 days. Seventeen patients (63.0%) used antibiotics within a week of FMT. In a culture-dependent method, the expression of VanA and overall genes significantly decreased (p = 0.011 and p = 0.003 respectively). In a culture-independent method, VanA, bla NDM, and overall gene expression significantly decreased over time after FMT (p = 0.047, p = 0.048, p = 0.002, respectively). Similar results were confirmed following comparison between each time point in both the culture-dependent and -independent methods. Regression analysis did not reveal important factors underlying the genetic response after FMT. No adverse events were observed. Conclusion FMT in patients infected with MDROs downregulates the expression of resistance genes, especially VanA, and facilitates MDRO decolonisation.