This study evaluates the effects of water quality, i.e. organic matter content, alkalinity, and pH, on acute copper toxicity to mosquitofish (Gambusia holbrooki Girard, 1859), an invasive species widespread in natural and artificial wetlands. The aim of this study was to improve our understanding of the consequences of CuSO4 treatments on biodiversity conservation in irrigation ponds. Tests were carried out using three water types: irrigation-pond water (IPW) of relatively high organic matter levels and slightly alkaline pH; IPW treated with sodium hypoclorite (IPW + SH) to remove organic matter and raise pH; dechlorinated tap water (DTW) with a relatively low alkalinity and organic matter, and a circumneutral pH. Fish were exposed for 96-h under static conditions to 0.25, 0.75, 1.5, and 4 mgCu/L. Mortality was monitored every 24 h and LC50 values were determined. After the 96 h period, the Cu concentration in gills was also determined. The LC50-96 h values were 1.90 (IPW), 1.51 (IPW + SH), and 0.78 (DTW) mg Cu/L, which can be considered relatively high. The concentration of copper in gills increased with the nominal Cu concentration in the three water treatments, and was significantly higher in DTW compared with IPW and IPW + SH. These results indicate that the relatively high alkalinity and, particularly, organic matter levels in pond water substantially buffer copper bioavailability to mosquitofish. However, chronic exposure to continuous Cu treatments and its accumulation in pond sediments are likely to produce stress symptoms at the individual and population levels of vertebrate and invertebrate species, which, besides agronomical and human health reasons, advise against using CuSO4 as a management tool for irrigation ponds.