There is a growing concern regarding the adverse effects of pharmaceutical pollution on aquatic environments. The present study investigated the toxicity of different antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs) on two green algae namely Chlorella sp. and Desmodesmus spinosus. Based on the 96h IC₅₀ values of the two chlorophytes, tetracycline (TET) was more toxic than other antibiotics (ciprofloxacin (CPF) and amoxicillin (AMX)), while paracetamol (PAR) was more toxic than ketoprofen (KET) and diclofenac (DIF). Gross photosynthesis was markedly reduced with most of the investigated drugs, although, the Chl a content was stimulated in a dose-dependent manner. Algal treated cells exhibited elevated malonaldehyde content which reflected several structural and functional cellular damages. Catalase and ascorbate peroxidase (APX) were involved in the reduction of reactive oxygen species, but the effects of APX were more pronounced at low drug concentrations. Box–Behnken design (BBD) was employed to investigate the combined toxicities of PAR, KET, AMX and TET on D. spinosus in response to growth inhibition and pigment increase. The mutual interactions varied between synergism and antagonism. The BBD analysis indicated that the experimental design could be effectively utilized as a useful tool for ecotoxicological assessment.