The effects of a mixture of pharmaceuticals at environmentally relevant concentrations were studied on growth and transcriptional regulation in zebrafish liver (ZFL) cells. The mixture of 13 ingredients was assembled to mimic the association and low concentration (ng/l) of drugs as detected in the environment, and decidedly inhibited ZFL cells proliferation in vitro over a 72 h exposure time. Using an oligonucleotide DNA-microarray targeting 14,000 zebrafish transcripts, we profiled gene expression in ZFL cells treated with ecologically relevant levels of the drug mixture. Compared to unexposed controls, ZFL cells challenged with pharmaceuticals were characterised by transcriptional repression involving primary metabolism and regulation of the cell cycle. On the other hand, we observed upregulation of genes identifying protein kinase signalling pathways and DNA-repair mechanisms. Part of the identified transcripts could be associated with general toxicity, while others were possibly linked to the effects of specific pharmaceuticals. Drugs also caused overexpression of oestrogen receptor beta and the oestrogen responsive protein GREB1. The context drawn by our data highlights a similarity in the response to the drug mixture in fish and previously employed human cells, and further prioritise studies targeting the potential risks associated with the presence of pharmaceuticals in the environment.