Extended spectrum β-lactamases (ESBL)-producing Escherichia coli have emerged worldwide in animal husbandry and they were reported from different ecosystems. The purpose of this study was firstly, to investigate the presence of ESBL-producing E. coli in the gastrointestinal (GIT) and reproductive (RT) tracts of broiler breeding roosters, and secondly to study the impact of an ESBL-producing E. coli on artificially infected semen. A total of seventeen ESBL-producing E. coli strains were isolated from the gastrointestinal and reproductive tracts of nine broiler breeding roosters. All isolates were identified to the species level by API 20E system and MALDI-TOF, serotyped, and genetically characterized for ESBL production. Semen was artificially infected with E. coli ATCC25922 or with an ESBL-producing E. coli strain recovered from the reproductive tract. A computer aided semen analyzer (CASA) was used to compare different spermatozoa motility parameters in each sample. All ESBL-producing E. coli isolates could not be typed with the currently used sera and they were harboring a blaCTX-M gene alone or in combination with a blaTEM gene. The semen quality was notably less affected in samples infected with ESBL-producing E. coli strain compared to the control and sample infected with E. coli ATCC25922. The present study revealed that ESBL-producing E. coli can be isolated from both reproductive and digestive tracts of broiler breeding roosters. Contamination of the reproductive tract with ESBL-producing E. coli could lead to contamination of semen and could be an important factor in the dissemination of ESBL-producing E. coli in poultry.