Zoonotic diseases, as infectious agents transmitted from animals to humans, represent a significant public health challenge, particularly in the context of climate change, ecosystem disruptions, and the increasing interaction between humans, animals, and the environment. The One Health approach emphasizes the importance of a multidisciplinary strategy for preventing and controlling these illnesses by integrating the human, veterinary, and environmental health sectors. Laboratory diagnostics play a pivotal role in the early detection of zoonotic pathogens, surveillance of their distribution within populations and the environment, and evaluation of the efficacy of control measures. Notably, Lyme disease, tularemia, brucellosis, and legionellosis serve as key indicators of the interrelationship between human health and environmental conditions. Lyme disease, the most common vector-borne zoonosis in Europe; tularemia, a zoonosis with epidemic potential; brucellosis, a severe occupational zoonosis with significant economic consequences; and legionellosis, a waterborne disease model, all require rigorous monitoring using advanced microbiological, molecular, and serological methodologies. The integration of laboratory diagnostic data within a comprehensive surveillance framework facilitates a more profound understanding of the sources and transmission pathways of these infections, thereby providing a scientific basis for the timely implementation of disinfection, disinsection, and deratization (DDD) measures and the protection of public health. Integrated diagnostics lie at the core of the One Health paradigm, recognizing the inseparability of human, animal, and environmental health. The establishment of an effective early warning and control system for zoonotic diseases can only be achieved through coordinated, interdisciplinary surveillance and diagnostic efforts.