A 2 × 3 factorial arrangement study was conducted to evaluate 3 dosages of 2-nitro-1-propanol (
NP
; 0, 150, and 200 ppm) on intestinal health of birds with or without
Eimeria
challenge. A total of 432 thirteen-day-old male broiler chickens were randomly allocated to 6 treatments with 8 replicate cages of 9 birds per cage. All birds were fed with treatment diets from day 13 to 21. Birds in the challenge groups were gavaged with
Eimeria maxima
(50,000 oocysts per bird),
Eimeria tenella
(50,000 oocysts per bird), and
Eimeria acervulina
(250,000 oocysts per bird) on day 15. Growth performance was evaluated from day 13 to 21, and gut permeability was measured by fluorescein isothiocyanate dextran on day 20. The intestinal lesion, intestinal morphology, and oocysts shedding were determined at the end of the trial. The linear and quadratic orthogonal polynomial contrasts were used to evaluate the effects of increasing NP doses in responses to
Eimeria
challenge. The results showed that NP was not able to maintain efficient growth performance but improved gut leakage during
Eimeria
infection period. On the other hand,
Eimeria
infection increased gut permeability (
P
< 0.0001) and reduced ileal digestible energy (
IDE
) and apparent ileal digestibility (
AID
) of nitrogen. However, the increase of NP linearly enhanced IDE and AID of nitrogen (
P
< 0.01). Moreover, an interaction between challenge and linear dosage effects was observed for IDE (
P
= 0.0066) and AID of nitrogen (
P
= 0.0462). The results indicated that NP improved nutrient digestibility and reduced total oocysts shedding in birds challenged with
Eimeria
spp. Besides, higher NP doses numerically improved villi height in the intestine. In summary, NP was not able to maintain growth performance of birds but presented positive outcomes on nutrient digestibility and reduced oocysts shedding during mixed
Eimeria
infection.