Introduction and Objective. Lyme borreliosis (LB) is the most frequent tick-borne disease with 17,338 cases reported in Poland in 2022. Since research on a LB vaccine is still ongoing, the promotion of individual behaviours and limiting of tick exposure is one of the most effective ways to prevent LB. The aim of the study was to evaluate the effectiveness of the LB prevention programme by assessing the knowledge, practice behaviours, seroprevalence of LB and satisfaction among the population of the Lublin Province in eastern Poland. Materials and method. The prevention programme was carried out among 2,920 participants who were asked about their exposure to ticks, history of LB and prevention behaviours. Awareness of knowledge was evaluated before and after training. Seroprevalence of LB was rated by ELISA and immunoblot assays. Results. Over 73% of participants reported tick bites in their lifetime, without significant differences between rural and urban area inhabitants. More than 80% of individuals declared that they use protective measures (PPM), such as proper clothes and body checking; repellents were the least frequently used by participants. The diagnosis of LB but not tick bites in a lifetime influenced the more frequent use of PPM. Increase in knowledge was observed in 86% of participants after education, and the highest knowledge was noted among respondents with higher education. The seroprevalence of anti-B. burgdorferi antibodies was 37% and was higher among men than women (40% vs. 36%). Conclusions. The population of Lublin Province is highly exposed to tick bites and infection with B. burgdorferi. The high seroprevalence and increase in knowledge confirmed the effectiveness and need for preventive programmes. These results can be useful for optimizing and enhancing the effects of future prevention campaigns.