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The aim of the study was to evaluate the bio-efficacy of two different acaricides against mobile stages of hard ticks Ixodes ricinus, Dermacentor marginatus, and Haemaphysalis punctata in their natural habitats. The study was conducted during 2020 and 2021 at localities populated by I. ricinus as the predominant species, at which the presence of Borrelia afzelii, Borrelia garinii, and Borrelia lusitaniae was confirmed. During the first investigation year, a combination of two pyrethroids, permethrin, and tetramethrin, with an insecticide synergist piperonyl butoxide (trade name: Perme Plus®) was tested. At the first evaluation, 24 h after the treatment with Perme Plus®, the efficacy expressed as a reduction rate of the population density was within the interval of satisfying performance (70-90%) at all localities, while the highest efficacy (97.8%) was recorded on the 14th post-treatment day. In the second investigation year, the formulation based on lambda-cyhalothrin (trade name: Icon® 10CS) was used. On the first post-treatment evaluation day, satisfying effects were also demonstrated. The highest recorded efficacy rate of lambda-cyhalothrin (94.7%) was recorded on the 14th post-treatment day. Both tested acaricides manifested satisfying initial acaricidal effects against mobile stages of ticks and provided long-term effects. Comparison of the regression trend lines of population reduction revealed that satisfying effects of treatment with Perme Plus® lasted until the 17th post-treatment day, while in the case of Icon® 10CS, the residual effects were significantly prolonged (30 days)., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)