Background: Tick-borne infections resulting from regular tick infestation in dogs are a common veterinary health problem all over the world. The application of repellent and acaricidal agents to prevent transmission of pathogens is a major protection strategy and has been proven to be highly effective in several trials under laboratory and natural conditions in dogs. Despite such promising results, many dog owners still report tick infestation in their dogs although acaricidal agents are used. Information about the current infection status and changes of the infection status regarding tick-borne diseases (TBD) in dogs treated by the owner’s controlled acaricide application is lacking. Methods: In this study 30 dogs were each treated with permethrin, fipronil + S-methoprene, or served as untreated controls. Application of the acaricide was performed by the owner who decided when and how often to use the spot on preparation. Over a period of 11 months, dogs were clinically examined and sampled for antibody responses against Babesia canis, Anaplasma phagocytophilum, Borrelia burgdorferi s. l., and TBE virus before the study started, 6 months later and at the end of the investigation period. Results: The permethrin acaricide was applied on average 3.40 times within the examination period, whereas the fipronil + S-methoprene medication was applied 3.03 times. Approximately 2/3 of all dogs, independent of the group, had a positive immune response to one or more pathogens. Three dogs developed clinical symptoms of canine babesiosis, all other dogs remained healthy. Individual number of ticks per dog or number of infections per dog did not correlate with the application rate, and the number of ticks per dog did not influence the number of infections per dog. As owners did not apply the acaricides regularly no influence on the number of infections could be documented although the number of ticks was clearly reduced by the application of the spot-on drugs. Conclusions: Clinical disease in dogs exposed to tick-borne pathogens is rare, although a humoral immune response reflecting infection is common. More educational training for dog owners is necessary to make the application of acaricides effective regarding the prevention of tick-borne diseases.