Urbanization imposes multiple anthropogenic stressors on freshwater ecosystems, affecting aquatic species physiological responses. This study explores the transcriptomic responses of the freshwater fish Cottus rhenanus to various stressors in an urban river system. RNA sequencing of fish from multiple stations revealed significant seasonal variations in gene expression, with a higher number of differentially expressed genes (DEGs) observed in the summer. Fish at the station experiencing the highest anthropogenic pressure showed notable responses, particularly during warmer months, with enriched pathways related to metabolism, oxidative stress, and immune responses. Key findings include the activation of metabolic stress pathways and immune system genes, such as IL-17 and MAPK pathways, influenced by high temperatures, salinity, and low oxygen levels. Pathway enrichment analyses highlight the impact of temperature and salinity on oxidative stress and osmoregulation, revealing the critical role of the transportome in adapting to salinity changes. These findings showcase the complex interactions between stressors and physiological responses, emphasizing the need for integrated conservation strategies to manage urban stream ecosystems.