The poultry red mite (PRM), Dermanyssus gallinae, a potential vector of pathogens to animals and humans, causes impaired bird welfare. A study investigated changes in behavioural variables, physiological biomarkers, and health parameters following acaricidal treatment of PRM infestation of laying hens on a commercial farm. Mite traps determined the challenge to 12,700 hens before and after drinking water administration of the acaricide, fluralaner (Exzolt®, 0.5 mg/kg; Weeks 0 and 1). Weekly daytime direct observations and night-time video recordings monitored bird behaviours from Weeks -6 through +6. Blood samples were collected from randomly-selected birds (Weeks -6, -1, and +6). Following treatment, mite count reductions (>99%) were statistically significant (P < 0.0001), as were night-time reductions in the percent of hens showing activity, preening, head scratching (all P < 0.0001), and head shaking (P = 0.0007). Significant daytime reductions were observed in preening and head scratching (both P < 0.0001), head shaking (P = 0.0389), severe feather pecking (P = 0.0002), and aggressive behaviour (P = 0.0165). Post-treatment, comb wounds were significantly reduced (P = 0.0127), and comb colour was significantly improved (P < 0.0001). Heterophil/lymphocyte ratio was significantly reduced at Weeks 1 and 6 (P = 0.0009 and P < 0.0001, respectively). At Week 6, blood corticosterone (P = 0.0041) and total oxidant status (P < 0.0001) were significantly reduced, and haemoglobin and mean corpuscular haemoglobin significantly increased (P < 0.0001). Farm production records indicated that those post-treatment improvements were accompanied by significant reductions in weekly mortality rate (P = 0.0169), and significant recovery in mean weekly egg weights (P < 0.0001) and laying rate (P < 0.0001). The improvements in behavioural variables, physiological biomarkers, and health parameters that were observed following the elimination of PRM on a commercial farm indicate that infestations can be a cause of reduced hen welfare.