Toxicity of antibacterial agents on environmentally relevant bacteria was investigated using activated sludge. The growth and nitrifying inhibiting effects for activated sludge of benzyl penicillin (penicillin G) (BP), tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC), olaquindox (O), streptomycin (ST), tiamulin (TI), tylosin (TYL) sulfadiazine (SDZ), metronidazole (MET), and oxolinic acid (OXA) was investigated. Studies were performed in accordance to the ISO 15522 (1999) and ISO 9509 (1989) test guidelines, respectively. The toxicity (EC50 value, mg/L) found with the ISO 15522 was in decreasing EC50 values; O (95.7), BP (84.6), TYL (54.7), TI (14.3), TC (2.2), OTC (1.2), ST (0.47), CTC (0.40), and OXA (0.1). No observed effect concentrations (NOECs) (mg/L) of 100 and 60, respectively was found for MET and SDZ. Triplicate tests assessing the effects of the antibacterial agents on the nitrification rate gave indications only as the level of increased or decreased rate. More accurate data for the inhibition of Nitrosomonas europaea was found with a suspended culture of the nitrifying bacteria. The toxicity (EC50 value, mg/L) found was in decreasing EC50 values; TI (23.3), SDZ (17.0), TC (4.0), OTC (1.7), OXA (1.0), CTC (0.64), O (0.03), ST (0.02). For MET and TYL, NOECs (mg/L) of 100 and 50 were found, respectively. The antibacterial agents were also assessed using a pour plate method with both (separately tested) activated sludge bacteria and N. europeae showing to be 5 to 10 times more potent to most agents except SDZ, TI, and MET.