Application of municipal biosolids to agricultural soil can improve soil quality and improve crop yields. However, runoff or tile leachate from biosolids-applied fields may contribute to localized eutrophication of surface water. A laboratory experiment was conducted to determine loss potential of nutrients from soils amended with two different biosolids (anaerobically digested and chemically stabilized) relative to loss from a reference soil and to determine response in freshwater microcosms to nutrients lost from soils. Total phosphorus (TP) and total nitrogen (TN) were measured in runoff, and equivalent amounts were added to reference microcosms to determine if aquatic systems would respond similarly to TN and TP loading in bioavailable forms (PO, NH, NO) simulating loading related to inorganic fertilizer application. Nutrient concentrations (TP, TN, PO, NH, NO, and organic P and N) were similar in the runoff from the two biosolids-amended soils and higher than those in the runoff from the reference soil. Runoff from biosolids-amended soils stimulated algal growth and production (chlorophyll a and dissolved oxygen) relative to runoff from reference soil, but the response was weaker than in microcosms receiving equivalent amounts of inorganic N and P. Nutrient runoff from land-applied biosolids does have potential to increase algal production in receiving waters; however, this experiment suggests receiving waters may absorb a single large nutrient loading event associated with runoff from biosolids-amended soil without substantial impact. Moreover, the response to N and P in biosolids versus inorganic nutrient additions suggests biosolids may contribute relatively less to eutrophication than inorganic fertilizers, assuming equivalent TN and TP loading to aquatic systems.