Dissolved organic matter (DOM) plays a significant role in protecting freshwater organisms against metal toxicity. To study this, reverse osmosis (RO) has been widely used as a highly efficient method for rapid collection of large quantities of DOM from natural surface waters. The objective of this study was to examine the potential impact of the RO isolation technique on the protective effects of DOM on the toxicity of copper and zinc to the cladoceran Daphnia magna and the green alga Pseudokirchneriella subcapitata. DOM was concentrated from a natural surface water using RO and at the same time a natural (unconcentrated) surface water was taken. The concentrated DOM was rediluted to the level of the natural water to obtain the so-called reconstituted water. Chemical analyses and toxicity tests were performed with both the natural surface water and the reconstituted water. First, most chemical parameters were not significantly changed by the RO sampling. For both copper and zinc, no significant differences were observed in 48 h-EC50s for D. magna and in 72 h-EC50s for P. subcapitata between the reconstituted water and the natural water. Hence, it may be concluded that reverse osmosis does not significantly affect the protective effect of natural DOM against copper and zinc toxicity.