This study was conducted to evaluate the effects of different dietary inclusion of eucalyptus essential oil (
EEO
) on growth performance, relative organ weight, cecal microflora, nutrient digestibility, serum biochemical parameters, and thigh muscle fatty acid profile in broiler chickens. A total of six hundred 1-day-old male broiler chickens were randomly allocated into 5 treatment groups with 8 replicate pens, and each pen contained 15 birds. The experiment lasted for 42 d. Dietary treatments included corn–soybean meal-based diet supplemented with 0, 250, 500, 750, and 1,000 mg/kg EEO. The results indicated that dietary treatments had no effect on growth performance parameters in the 1 to 10 d period. From day 11 to 24, dietary supplementation of EEO showed a linear decrease in feed conversion ratio (
FCR
,
P
< 0.05). From day 25 to 42 and the overall period (1–42 d), broilers fed with different levels of EEO showed a linear increase in body weight gain (
BWG
) and reduction in feed conversion ratio (linear,
P
< 0.05). The relative organ weight were unaffected by any of the dietary treatments. With increasing levels of EEO, the cecal
Escherichia coli
(linear,
P
= 0.085) count showed a trend in reduction, and the cecal lactic acid bacteria population tended to increase (linear,
P
= 0.063). The apparent ileal digestibility of ether extract and organic matter were linearly and quadratically increased in response to increasing dietary EEO supplementation (
P
< 0.05). A trend of linear decrease in total cholesterol in the serum of birds fed with different levels of EEO was recorded (
P
= 0.074). Eucalyptus essential oil's inclusion increased serum superoxide dismutase linearly but reduced serum malondialdehyde linearly (
P
< 0.05). Dietary supplementation of EEO affected the fatty acid profile of thigh muscle so that increased the concentrations of total polyunsaturated fatty acids (linear,
P
< 0.05) and reduced total saturated fatty acid contents (linear,
P
< 0.05). Taken together, the inclusion of EEO increased BWG and decreased FCR during day 25 to 42 and day 1 to 42, and partially improved cecal microflora balance, nutrient digestibility, antioxidant activity, and thigh muscle fatty acid profile in broiler chickens.