Dermanyssus gallinae (Mesostigmata: Dermanyssidae) is the most harmful ectoparasite of laying hens, represents an occupational hazard for poultry workers, and a growing threat to medical science per se. There is increasing demand for alternative products, including plant-derived acaricides, with which to control the mite. The present study investigated the efficacy of neem oil against D. gallinae on a heavily infested commercial laying hen farm. A novel formulation of 20% neem oil, diluted from a 2400-p.p.m. azadirachtin-concentrated stock (RP03™), was administered by nebulization three times in 1 week. Using corrugated cardboard traps, mite density was monitored before, during and after treatment and results were statistically analysed. Mite populations in the treated block showed 94.65%, 99.64% and 99.80% reductions after the first, second and third product administrations, respectively. The rate of reduction of the mite population was significantly higher in the treated block (P < 0.001) compared with the control and buffer blocks. The results suggest the strong bioactivity of neem, and specifically of the patented neem-based formulation RP03™, against D. gallinae. The treatment was most effective in the 10 days following the first application and its effects persisted for over 2 months. Further studies will aim to overcome observed side effects of treatment represented by an oily layer on equipment and eggs.