Triclosan (TCS) antimicrobial compound is used in personal care products such as shampoos and soaps. The TCS compound can survive the treatment process and is often present in the sewage effluent and biosolids. We investigated the impact of TCS upon selected microbiological and biochemical parameters in two contrasting Australian soils. Substrate-induced respiration and nitrification, plus activities of four enzymes (relevant for carbon and nitrogen turnover), were measured using Organisation for Economic Co-operation and Development protocols after exposing soils to TCS at concentrations of 0, 1, 5, 10, 50, and 100 mg/kg of soil. Respiration in the sandy soil was not affected by the addition of TCS, but in clay soil it decreased to TCS at 50 mg/kg of soil. The nitrification process was affected in both soils. In the sandy soil, TCS showed a negative effect on the nitrate and nitrite production at 5 mg/kg. In contrast, in the clay soil, the effect was noticeable only at concentrations greater than 50 mg/kg. The response of four enzymes (acid and alkali phosphatase, 3-glucosidase, and chitinase) in the two soils was variable, and only P3-glucosidase showed some response to TCS addition. The study demonstrated that TCS at concentrations below 10 mg/kg can disturb the nitrogen cycle in some soils. A first-tier hazard assessment highlighted the need for further work on TCS impact on soil microorganisms.