Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae are classified as serious threats to human health by the U.S. Centers for Disease Control and Prevention. Water used for irrigation of fresh produce can transmit such resistant bacteria directly to edible plant parts. We screened ESBL-producing Escherichia coli, Enterobacter cloacae, and Citrobacter freundii isolated from irrigation water, for their potential to transmit resistance to antibiotic-susceptible E. coli. All strains were genome-sequenced and tested in vitro for transmission of resistance to third generation cephalosporins on solid agar as well as in liquid culture. Of the 19 screened isolates, five ESBL-producing E. coli were able to transfer resistance with different efficiency to susceptible recipient E. coli. Transconjugant strains were sequenced for detection of transferred antibiotic resistance genes (ARG) and compared to the known ARG pattern of their respective donors. Additionally, phenotypic resistance patterns were obtained for both transconjugant and corresponding donor strains, confirming ESBL-producing phenotype of all obtained transconjugants.