This study investigated the seasonal and spatial dynamics of off-flavour compounds—geosmin and 2-methylisoborneol (2-MIB)—in an intensive rainbow trout (Oncorhynchus mykiss) aquaculture system for human consumption in western Spain. Weekly water and fish flesh samples were collected over a 12-month period from three farms supplied by the River Tormes. Physicochemical parameters, determination of geosmin and 2-MIB by SPME-GC-MS, microbial counts, and microbial community composition were assessed alongside volatile compound concentrations. Geosmin and 2-MIB showed marked seasonal variation, with peak levels in water and fish flesh during spring and summer, correlating positively with temperature. Geosmin accumulation in fish was highest in the downstream farm, suggesting cumulative exposure effects. In contrast, 2-MIB was detected only in water and at lower concentrations. Microbial analyses revealed high bacterial and fungal diversity, including cyanobacterial taxa such as Phormidium setchellianum and Pseudoanabaena minima, known producers of geosmin and 2-MIB. These findings highlight the importance of water microbiota and environmental conditions in off-flavour development. Managing cyanobacterial populations and monitoring spatial-temporal variability are essential to mitigate the development of earthy or musty flavours and economic losses in aquaculture systems.