The emergence of multidrug-resistant bacteria has become a major problem. Cockroaches may play an important role in the spread of those bacteria between the environment and humans. This study was designed to screen extended-spectrum β-lactamase (ESBL)-producing and colistin-resistant strains and to investigate the molecular support of multidrug-resistant in the external surface and gut homogenates of cockroaches collected from different locations in Tunisia. Between July 2017 and June 2018, 144 samples were isolated from 115 trapped cockroaches (collective catering, houses, and a hospital). Antibiotic susceptibility testing was performed using the disk diffusion method. Extended-spectrum β-lactamase-encoding genes and the gene were investigated by real-time PCR (RT-PCR) and standard PCR. The genetic relationship among isolates was studied with the help of multilocus sequence type (MLST) analysis. Of the 144 isolates, 22 strains exhibited a positive ESBL-screening test (73.3%), including 17 Escherichia coli isolates and 5 Klebsiella pneumoniae isolates. Among them, 9 Escherichia coli isolates were resistant to colistin, with an MIC ranging from 8 to16 μg/L, all of which harbored the gene. Eight genes were detected; two among them were associated with and , and seven genes were detected that also harbored the gene. Genotyping analysis revealed 7 different sequence types already described in humans and animals. We report the first survey of in ESBL-producing E. coli isolates from cockroaches. Our findings highlight cockroaches as a source of nosocomial infections, and they are a reservoir of colistin-resistant E. coli, which is a carrier of other additional risk genes such as , especially in hospitals. Multidrug resistance in has become a major concern worldwide that is increasingly observed in human, animals, and also cockroaches. In our study, we found that cockroaches may play an important role as a potential vector of multidrug-resistant in the hospital environment and collective catering. Our study describes the first survey of in ESBL-producing E. coli isolates from hospital cockroaches. Our results further highlight the possibility that may enter humans via cockroach contamination and thereby threaten public health. Our results show that these cockroaches are an important reservoir of colistin-resistant E. coli and carriers of other additional risk genes such as , hence the importance of strengthening prevention strategies and of strictly respecting hygiene measures in order to control their distribution and spread in Tunisia.