The aim of this study was to investigate the fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni. The in vitro growth, the survival on food matrix, and the in vivo colonization of C. jejuni and C. coli susceptible isolates and their isogenic resistant mutants were studied. In vitro experiments demonstrated that macrolide resistance imposed a fitness cost when the susceptible strains and their isogenic resistant mutants were cultured in competition. When inoculated in food matrix, the resistant C. jejuni mutant was no longer detectable after 3 to 5 days but the susceptible strain remained detectable for over 18 days. No difference in survival in food matrix was observed between susceptible and resistant C. coli. When inoculated in vivo in chickens, the macrolide susceptible and resistant C. coli displayed similar levels of colonization, both in separated inoculations and during competitive assays. Strikingly, when mono-inoculated or co-inoculated into chickens, macrolide susceptible C. jejuni outcompeted the macrolide resistant population. However, a spontaneous mutant that evolved in vivo showed a colonization capacity similar to the susceptible strain. Our findings demonstrate the effect of macrolide resistance on the fitness of Campylobacter but suggest that evolved mutants may be as fit as susceptible strains.