Simple SummaryCoccidiosis, which is induced byEimeriaspp., is one of the most predominant diseases and causes tremendous economic losses in the world. The effects ofEimeriainfection on broilers at slaughter ages in floor pen conditions should be elucidated to conduct further studies investigating the effects of feed additives and bioactive compounds as alternatives for anti-coccidial drugs in broilers. The study was aimed to investigate the effects of different inoculation doses ofE. acervulina,E. maxima, andE. tenellawith different doses on growth performance, gut ecosystem, and oocyst shedding in broilers raised in floor pens for 35 days.Eimeriainfection decreased body weight (BW) in the acute phase (D 21), and this effect was prolonged to the final day (D 35).Eimeriaoocysts were observed in the litter until D 35, which may indicate thatEimeriaspp. reinfected broilers.Eimeriainfection dramatically reduced crude fat (CF) digestibility in the acute phase, which may be associated with reduced fat content in the broilers on D 35. Gut microbiota was negatively affected byEimeriainfection in both acute phase and on D 35. In conclusion,Eimeriainfection negatively affected the growth performance and gut ecosystem in broilers, and the negative effects were prolonged to D 35 in floor pen conditions.AbstractThe study was conducted to investigate the effects of differentEimeriainoculation doses on the growth performance, gut ecosystem, and body composition of broilers in floor pens for 35 days. A total of 750 15-day-old broilers were allocated to five experimental groups with six replicate pens. The five experimental groups included unchallenged control (CON);Eimeriadose 1 (ED1):E. acervulina: 31,250/E. maxima: 6250/E. tenella: 6250;Eimeriadose 2 (ED2):E. acervulina: 62,500/E. maxima: 12,500/E. tenella: 12,500;Eimeriadose 3 (ED3):E. acervulina: 125,000/E. maxima: 25,000/E. tenella: 25,000; andEimeriadose 4 (ED4):E. acervulina: 250,000/E. maxima: 50,000/E. tenella: 50,000. On D 21, BW were linearly reduced by increasedEimeriainoculation doses (p< 0.01). On D 35, theEimeriachallenge groups had significantly lower BW compared to the CON group. IncreasedEimeriainoculation doses linearly decreased crude fat (CF) (p< 0.01) on D 21. IncreasedEimeriainoculation doses tended to increase the relative abundance of the phylum Proteobacteria (p= 0.098) on D 21. On D 35, lean:fat was linearly reduced by increasedEimeriainoculation doses (p< 0.05).Eimeriainfection negatively influenced growth performance and gut health in broilers in the acute phase, and the negative effects were prolonged to D 35 in floor pen conditions.