Sewage sludge, or biosolids, produced in wastewater treatment plants (WWTP) can be used as an alternative to organic fertilizer in the agricultural field. However, it holds a large amount of anthropogenic chemicals, such as drugs and metals. The aim of the present study is to evaluate biochemical biomarker responses in earthworms (Eisenia andrei) exposed to soil presenting different sludge concentrations deriving from the largest WWTP in Southern Brazil. Treatments comprised 3%, 6%, 12%, 24%, 50%, and 75% of sludge incorporated to native forest soil, 100% of sludge and the control group (0%). Concentrations of different drugs, mainly of antibiotics, as well as high sulfur, magnesium, potassium, zinc, and phosphorus were identified in analyzed sewage sludge. Exposed earthworms were collected on the 7th, 14th, and 28th day of the experiment and subjected to analysis of lipid peroxidation levels (TBARS), acetylcholinesterase (AChE), catalase (CAT), and glutathione S‐transferase (GST) activity. Treatments with 50%, 75%, and 100% of sludge were lethal to earthworms after 7 days. Based on biochemical tests performed in treatments with 3%, 6%, 12%, and 24% of sludge, the highest sludge concentrations (12% and 24%) and the longest exposure time (28 days) led to significant changes in biomarkers. Therefore, sewage sludge must be incorporated into the soil at concentrations lower than 3%, mainly due to changes in CAT, AChE, GST, and the resulting lipid damage observed at concentrations of 6%, 12%, and 24%.