The poultry red mite Dermanyssus gallinae is the most important blood-sucking ectoparasite in egg-laying hen facilities. The aim of this study was to evaluate the in vitro acaricidal activity of different concentration of authorized (phoxim, ByeMite®, 500 mg/mL) and unauthorized (fipronil, Frontline® 250 mg/100 mL spray) molecules on 14 field isolates of D. gallinae collected from different ornamental poultry farms from different Italian regions. The sensitivity test was performed by contact exposure to four concentrations of each insecticide diluted at 1:5 (10,000-2000-400-80 ppm for phoxim, 500-100-20-4 ppm for fipronil) on a filter paper. The effectiveness of the treatment was significantly (p < 0.0001) associated with the dose of the pesticide used. Considering the mean lethality, phoxim has greater efficacy compared to fipronil (p < 0.001). A great variability of lethality rate was observed with the increase in fipronil dilution; conversely, for phoxim, some outliers were observed, particularly in one farm, suggesting the hypothesis that a certain degree of resistance in the mite population could occur possibly as a consequence of the continual contact with the molecule. This underlines the importance of the use of licensed products administered at correct dosages and the need for alternative molecules to avoid the onset of drug resistance phenomena.