The Double Mountain Fork Brazos River (Texas, USA) consists of North (NF) and South Forks (SF). The NF receives urban runoff and twice-reclaimed wastewater effluent, whereas the SF flows through primarily rural areas. The objective of this study was to determine and compare associations between standard water quality variables and ichthyotoxicity at a landscape scale that included urban (NF) and rural (SF) sites. Five NF and three SF sites were sampled quarterly from March 2008 to March 2009 for specific conductance, salinity, hardness, pH, temperature, and turbidity; and a zebrafish (Danio rerio) embryo bioassay was used to determine ichthyotoxicity. Metal and nutrient concentrations at all sites were also measured in addition to standard water quality variables in spring 2009. Principal component analyses identified hardness, specific conductance, and salinity as the water variables that best differentiate the urban NF (higher levels) from rural SF habitat. Nutrient levels were also higher in the NF, but no landscape scale patterns in metal concentrations were observed. Ichthyotoxicity was generally higher in NF water especially in winter, and multiple regression analyses suggested a positive association between water hardness and ichthyotoxicity. To test for the potential influence of the toxic golden alga (Prymnesium parvum) on overall ichthyotoxicity, a cofactor known to enhance golden alga toxin activity was used in the bioassays. Golden alga ichthyotoxicity was detected in the NF but not the SF, suggesting golden alga may have contributed to overall ichthyotoxicity in the urban but not in the rural system. In conclusion, the physicochemistry of the urban-influenced NF water was conducive to the expression of ichthyotoxicity and also point to water hardness as a novel factor influencing golden alga ichthyotoxicity in surface waters.