Emerging contaminants (ECs) are not monitored nor regulated consistently, but may have negative effects on human health and ecosystem balance. Although pharmaceuticals and personal care products are among the main ECs found in surface and wastewater, their toxicity and fate are currently not sufficiently studied. In this study, we analyzed for the first time a group of 46 ECs in the secondary effluent of the wastewater treatment plants (WWTP) of Prague. Thirty-seven compounds were identified in the discharge to surface water. Three compounds had no toxicology information on Artemia salina: furosemide, hydrochlorothiazide, and tramadol. We performed acute toxicity (LC50) tests and enzyme assays after 24 and 48 h at room temperature and 28 °C for these three compounds. LC50 ranged from 225.01 mg/L for furosemide, the most toxic, up to above 14,000 mg/L for tramadol. Changes in enzymatic activity for GST, GPx, AChE, and LDH when A. salina were exposed to LC25 for each contaminant were conspicuous and significant in a contaminant-, exposure time-, and temperature-dependent manner. These biochemical markers complement the toxicity profile of these contaminants in aquatic ecosystems and highlight the need for further research on other ECs and their implications, and the regulations required to protect human and ecological health.