With the goal of assessing the environmental risk of pharmaceuticals, we have previously observed that a mixture of 13 different drugs at environmentally relevant concentrations had adverse consequences on human and zebra fish cells in vitro. Here we aimed to identify both main and interaction effects within the same environmentally relevant mixture of pharmaceuticals. We studied in vitro cytotoxicity in Escherichia coli, human embryonic HEK293, and estrogen-responsive OVCAR3 tumor cells using fractional-factorial experimental design. Our approach identified a subset of compounds of primary environmental concern, namely atenolol, bezafibrate, ciprofloxacin, and lincomycin, that had statistically significant effects on prokaryotic and eukaryotic cells at environmentally relevant exposure levels (ng/l). Drugs could interact and behave as chemosensitizers, with joint effects representing a statistically significant element of mixture toxicity. Effects and interactions were concentration dependent, confirming the difficulty of dose extrapolation in mixture toxicity data. This study suggests that a thorough investigation of mixture effects can direct environmental concerns toward a handful of pharmaceuticals, which may represent an actual risk at environmental concentrations. We indicate that risk identification may strongly depend on the use of environmentally relevant exposure scenarios. Antagonistic-synergistic interactions and dose dependency of effects may hamper the modeling and prediction of mixture toxicity with pharmaceuticals. Hazard identification for micropollutants depends heavily on appropriate study designs, and we indicate the use of in vitro cytotoxicity threshold and statistical design of experiments (DOEs) as a valid approach.