Dyes are widely used in various sectors and can be released into the environment where they persist for a long time because of their high stability to light or temperature and their resistance to environmental degradation. Dyes are often poorly characterized and toxicological/ecotoxicological data are available only for a few. These features, coupled with their toxicity, make dyes a possible source of ecological concern, particularly for fresh water aquatic ecosystems. Therefore, new data may be very useful for their risk assessment. In the present study, we investigated the aquatic toxicity of 42 commercial dye formulations using the application of in silico tools and ecological bioassays. The in silico approach was used to assess the similarities among the dyes, highlighting that dyes from the same chemical class are generally similar. No correlation was found among dyes with the same color. Acute and long-term ecotoxicological assays with daphnids and algae were applied to evaluate the potential impact of these products, according to the OECD guidelines 201 and 202. The bioassays were able to identify structures with potential ecotoxicity: only 9 formulations showed toxicity lower than 100 mg/L for daphnids while 30 dyes were toxic for algae. In our experimental conditions, algae were more sensitive to dye toxicity, particularly when the effects on cell number were considered.