Interstitial water conditions in the hyporheic zone of the stream bed are determinants of salmonid egg hatching success. We used standardised egg exposures to develop and validate discriminant analysis and generalised linear model models linking the hatching success of brown trout (Salmo trutta) with physicochemical factors of the interstitial zone (e.g., oxygen, specific conductance, nitrate, nitrite, ammonium, pH and redox potential). Interstitial water quality was identified as a limiting factor for egg development (median of relative hatching rates = 0.23). Hatching success was unimodal in hatchery and field references incubated in free-flowing water, but bimodal (very high or very low hatching success) in natural sediment exposures. The effects of physicochemical factors on the hatching success of Salmo trutta strongly depended on both the time and spatial scale analysed. The variables retained in the models differed between the macroscale (over all rivers), the river-specific scale (within a river) and the microscale (at different sediment depths). Egg hatching success decreased with increased substratum depth (decrease of 26% in 150 mm compared with 50 mm). Increasingly more variable interstitial water conditions (e.g., oxygen) throughout the egg incubation period suggest progressive degradation rates in the stream substratum during the incubation period at the microscale level. Consequently, consideration of different spatial and temporal scales is necessary for the evaluation of habitat quality in salmonid conservation and catchment management plans.