Water systems are significant but vulnerable components of karst areas. Our paper presents the results of a multidisciplinary approach focused on the identification of a whole complex of influences that can affect water quality in a karst area. We delineate results on the example of Jedovnicky brook, representing one of the main watercourses bringing water and, concurrently, pollution from the non-protected area out of karst to the Protected Landscape Area Moravian Karst (Czech Republic). The main aim is to present the development of human impacts on water quality in the period 1931 and 2010. The development of physical-chemical status (monitoring campaigns from 1949 to 2014; mass flows of BOD, NH₄-N, NO₃-N, and PO₄-P) as well as historical land use and demographic changes were assessed. Agricultural activities, population growth and changes in settlements were identified as the main sources of the pollution in the past. Agricultural activities have undergone major positive changes – especially in the way of management and the use of fertilizers. Currently, the strong suburbanization trend (rising population after the year 2000, the expanding built-up areas, increasing amount of municipal wastewaters) is the most important risk factor for the deterioration of Jedovnice brook water quality and, as a result, karst water quality.