Campylobacter is a major waterborne pathogen that can be found in rivers of the Mediterranean area. Characteristics of these rivers change throughout the seasons due to variations in environmental parameters. As these variations may affect water survival of Campylobacter, we analyzed it in the Llobregat River using three approaches whose complexity increase progressively: (i) river water microcosms in the laboratory subjected to varying temperatures; (ii) in situ experiments carried out in the river, in which bacteria were exposed to varying levels of environmental parameters; and (iii) monitoring of thermotolerant Campylobacter in the river over two years. Campylobacter was quantified using the most probable number (MPN) method. The results showed that an increase in water temperature accelerates Campylobacter inactivation, measured as the loss of culturability. In situ experiments revealed that inactivation rates were also affected by sunlight, but not by pH, oxygen concentration or water conductivity. These observations are supported by the seasonality detected in Llobregat River. Campylobacter inactivation was fastest in spring and summer, when temperature and solar radiation were at their highest. The results highlight the importance of considering the inactivation rates in natural conditions to improve the monitoring of this pathogen and thus evaluate properly the health risk associated to water.