Oomycete pathogens in freshwaters, such as Saprolegnia parasitica and Aphanomyces astaci, are responsible for fish/crayfish population declines in the wild and disease outbreaks in aquaculture. Although the formation of infectious zoospores in the laboratory can be triggered by washing their mycelium with natural water samples, the physico-chemical properties of the water that might promote sporulation are still unexplored. We washed the mycelia of A. astaci and S. parasitica with a range of natural water samples and observed differences in sporulation efficiency. The results of Partial Least Squares Regression (PLS-R) multivariate analysis showed that SAC (spectral absorption coefficient measured at 254 nm), DOC (dissolved organic carbon), ammonium-N and fluoride had the strongest positive effect on sporulation of S. parasitica, while sporulation of A. astaci was not significantly correlated with any of the analyzed parameters. In agreement with this, the addition of environmentally relevant concentrations of humic acid, an important contributor to SAC and DOC, to the water induced sporulation of S. parasitica but not of A. astaci. Overall, our results point to the differences in ecological requirements of these pathogens, but also present a starting point for optimizing laboratory protocols for the induction of sporulation.