Antimicrobial-resistant bacteria originating from broilers pose an ongoing challenge as they can spread in the environment and food chain. One approach to lower colonization is to administer live bacteria to broilers. A live Escherichia coli (E. coli) vaccine, consisting of a single E. coli strain used as a competitive exclusion (CE) approach, was evaluated to decrease the colonization with ESBL-/pAmpC- producing E. coli. 168 ESBL-/pAmpC- negative Ranger Gold broilers were divided into six groups (3 x n = 46 experimental groups; 3 x n = 10 control groups for quality assurance). The E. coli vaccine was administered on day one through coarse spray or by drinking water on day five. Experimental groups were orally co-colonized with 102 cfu of one ESBL- (ST410, blaCTX−M−15) and one pAmpC- producing E. coli (ST10, blaCMY−2/mcr-1) on day three. Colonization status was monitored throughout the trial and quantified at the end of the study (day 49). A transient reduction in ESBL-producing E. coli colonization (p < 0.001) was observed following coarse spray administration. However, this decrease was not sustained over time. It can be concluded that a single E. coli strain originating from a live vaccine cannot decrease colonization of broilers with ESBL-/pAmpC- E. coli throughout a fattening period.