Climate change has expanded tick habitats, leading to greater human exposure to ticks and the pathogens they carry, especially in areas where nature-preserving activities like reforestation and urban greenspaces create overlapping habitats. In Northern Europe, <i>Ixodes ricinus</i> is the most prevalent tick species, known for transmitting <i>Borrelia burgdorferi</i> s.l., the causative agent of Lyme borreliosis (LB). In the Netherlands, LB is the most common vector-borne disease, with its incidence quadrupling since the 1990s. Other known <i>I. ricinus</i>-borne pathogens, including <i>Anaplasma phagocytophilum</i>, <i>Babesia</i> species, and <i>Rickettsia helvetica</i>, are emerging. Little is known about their epidemiology, which requires further study. The dissertation has two main objectives: (i) evaluating the performance of commercially available antibody assays and test strategies for LB diagnosis and their clinical interpretation, and (ii) assessing the prevalence of human antibodies against tick-borne pathogens and associated risk factors in various populations. The main conclusions of part I of this dissertation were that commercially available assays and test strategies perform comparably well. While IgG or combined IgM/IgG assays and strategies showed high sensitivity and acceptable specificity, the added value of IgM testing is debatable. Despite advancements in serological testing, a reliable marker for active LB is still lacking, emphasizing the importance of interpreting laboratory results in the context of clinical history. A survey among medical microbiologists in the Netherlands revealed challenges in diagnosing active disease for low-incidence Lyme manifestations, suggesting the need for continued education. Artificial intelligence-guided algorithms and a case register could aid clinical decision-making, resulting in optimized diagnostic accuracy and increased patient trust. In part II, a study of 2016/2017 data found a 4.4% prevalence of <i>B. burgdorferi</i> s.l.-specific antibodies in the general population, with higher rates in men, older individuals, and those with frequent tick bites. Comparisons with 1995/1996 data showed a significant increase in seroprevalence over time, particularly among those over 50, possibly due to increased outdoor activities. A cohort of nature management workers showed higher seropositivity rates for <i>B. burgdorferi</i> s.l. and demonstrated exposure to non-borrelial pathogens, indicating occupational exposure risks. Future research should focus on identifying reliable markers for active LB and developing state-of-the-art diagnostics for rare pathogens. Continued surveillance and diagnostic development are crucial for understanding the epidemiology and pathology of tick-borne diseases, ultimately aiding in disease prevention and burden reduction.