Aim:This study aimed to estimate the prevalence, to characterize, and to evaluate antimicrobial susceptibility of pathogenic E. coli and Salmonella spp.in stray cats and dogs. Methods:Between November 2019 and October 2020, 199 faecal samples were obtained from 102 cats and 97 dogs rescued by a kennel in Lisboa. Isolation was performed in selective and non-selective medium, followed by identification of virulence genes of E. coli by PCR, and Salmonella serotyping. Antimicrobial susceptibility testing (AST) was performed according to EUCAST. mcr-1-5 genes were detected by PCR. All pathogenic isolates were further characterized by WGS. Results:The overall rate of E.coli and Salmonella was 73.9% (147/199) and 0.5% (1/199), respectively. Pathogenic E. coli were detected in 19.6% dogs and 18.6% cats. No cases of STEC were found. AST revealed that 13.0% of the isolates were resistant (16.2% dogs vs. 10.0% cats). Resistance to ampicillin, tetracycline, sulfamethoxazole, and/or trimethoprimwere the most frequent. MDR was observed in four ETEC isolated from dogs, which have the same ST155 and blaTEM-1B, sul1, sul2, dfrA1, and tet(A) resistance genes. All commensal E. coli were susceptible to antimicrobials. Salmonella (S.4,5:i:-) was identified in a cat. Belongs to ST3478 and presents ac(6')-Iaa, blaTEM-1B, and sul2 resistance genes. None isolate was ESBL or harboured mcr genes. Conclusions:Some of the isolates have relevant antimicrobial resistance. Due to the close contact with humans and to the potential release of pathogenic and resistant enteric bacteria in the environment, it is important to monitor stray cats and dogs health status following a One Health approach. Funding:This work is part of DiSCoVeR project (JRP24-R2-FBZ2-DiSCoVeR), funded by the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No. 773830.