The northern fowl mite (Ornithonyssus sylviarum) is a key pest for caged laying hens. High infestation levels can lead to anemia, reduced production, and mortality. Although the mites cause skin irritation and thus likely affect resting behavior, the nocturnal behavior of infested chickens has not yet been studied. We investigated the impact of mite infestation on nighttime behavior of 16 beak-trimmed White Leghorn hens. The hens were housed individually in wire cages. They were experimentally inoculated with approximately 35 mites at 25 weeks of age and observed for the behaviors dozing, sleeping, preening and being active. Continuous observations were made from video recordings taken from 22:00 h until 06:00 h for two consecutive nights at pre-infestation week 0 and post-infestation weeks 3, 5 and 7. Mite infestation levels were measured weekly on an 8-point scale (0 (no mites) to 7 (≥10,000 mites per hen)). For statistical analyses with linear mixed models, mite infestation levels were categorized as score 0 (no mites), 3–4 (51–500), 5 (501–1000) and 6–7 (>1000). Higher levels of mite infestation were associated with hens spending less time dozing (P < 0.001) and more time preening (P < 0.001) and being active (P = 0.012). Dozing decreased from 90% of the observed time for mite score 0 to 76% for score 6–7. Preening increased from 2% (score 0) to 9% (6–7) and time spent being active increased from 1% (0) to 7% (6–7). Mite infestation increased the number of uninterrupted bouts of all behaviors (P ≤ 0.020), especially dozing and preening (P < 0.001), suggesting restlessness in the mite-infested hens due to irritation and itching. The mite-infested hens’ increased nocturnal activity, including preening, as well as the fragmentation of behavioral activities together with decreased dozing, indicate disturbed resting behavior and suggest a reduction in the welfare of mite-infested hens.