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Hospital environments constitute the main reservoir of multidrug-resistant bacteria. In this study we aimed to investigate the presence of Gram-negative bacteria in one Northwestern Tunisian hospital environment, and characterize the genes involved in bacterial resistance. A total of 152 environmental isolates were collected from various surfaces and isolated using MacConkey medium supplemented with cefotaxime or imipenem, with 81 fermenter bacteria (27 <i>Escherichia coli,</i> and 54 <i>Enterobacter</i> spp., including 46 <i>Enterobacter cloacae</i>), and 71 non-fermenting bacteria (69 <i>Pseudomonas</i> spp., including 54 <i>Pseudomonas aeruginosa,</i> and 2 <i>Stenotrophomonas maltophilia</i>) being identified by the MALDI-TOF-MS method. Antibiotic susceptibility testing was performed by disk diffusion method and E-Test was used to determine MICs for imipenem. Several genes implicated in beta-lactams resistance were characterized by PCR and sequencing. Carbapenem resistance was detected among 12 isolates; nine <i>E. coli</i> (<i>bla</i>_NDM-1 (<i>n</i> = 8); <i>bla</i>_NDM-1 + <i>bla</i>_VIM-2 (<i>n</i> = 1)) and three <i>P. aeruginosa</i> were carbapenem-resistant by loss of OprD porin. The whole-genome sequencing of <i>P. aeruginosa</i> 97H was determined using Illumina MiSeq sequencer, typed ST285, and harbored <i>bla</i>_OXA-494. Other genes were also detected, notably <i>bla</i>_TEM (<i>n</i> = 23), <i>bla</i>_CTX-M-1 (<i>n</i> = 10) and <i>bla</i>_CTX-M-9 (<i>n</i> = 6). These new epidemiological data imposed new surveillance strategies and strict hygiene rules to decrease the spread of multidrug-resistant bacteria in this area.