Background: Whether affective states acutely predict the hypothalamic–pituitary–adrenal (HPA) axis activities and whether energy balance-related behaviors moderate the affect–HPA axis relationship in obese youths are not well-understood. Methods: 87 mostly obese (94.3% obese) minority adolescents (mean: 16.3 ± 1.2 years old; 56.8% Latino and 43.2% African American) participated in a randomized crossover trial in an observation laboratory, where they received either high-sugar/low-fiber (HSLF) or low-sugar/high-fiber (LSHF) meals first and then crossed over in the next visit 2–4 weeks later. During each visit, they rated five affective states and provided a saliva sample every 30 min for the first 5 h and wore a waist-worn accelerometer. The association between the affect ratings and cortisol levels in the subsequent 30 min and the moderation effect of energy balance-related behavior were examined using multilevel models. Results: Within-person negative affect (β = 0.02, p = 0.0343) and feeling of panic (β = 0.007, p = 0.004) were acutely related to the subsequent cortisol level only during the HSLF condition. The time spent in moderate-to-vigorous physical activity did not moderate the acute relationship between affect and the subsequent cortisol level. Conclusions: Negative affect could be acutely related to heightened HPA axis activities in youths, but only when they were exposed to meals with high sugar and low fiber content. These results suggest that the meals’ sugar and fiber content may modulate HPA axis reactivity to negative affect in youths.