Treated wastewater (TWW) reuse for irrigation has become an excellent way to palliate water scarcity in Mediterranean arid regions. However, the toxicological effects of these effluents on the soil's organisms, especially earthworms, have not been well studied as yet. In this paper, earthworms Eisenia andrei were exposed for 7 days and 14 days to five agricultural soils irrigated with TWW for different periods: 1 year, 8 years, and 20 years. In addition, they were also exposed to soil from one reference site sampled from the Ouardenin perimeter in the Monastir Governorate in Tunisia. The effect on earthworms was assessed at the biochemical level by evaluating for catalase (CAT), glutathione-S-transferase (GST), malondialdehyde accumulation (MDA) and acetylcholinesterase inhibition (AChE). On the other hand, genotoxicity and transcriptomic responses were evaluated using micronuclei test (MNT) and gene expression level of CAT and GST. Moreover, metals uptake by earthworms was analyzed. Results showed that CAT and GST activity in the earthworm increased significantly when they were exposed to soils irrigated with TWW for 1, 8 and 20 years. Furthermore, MDA concentration also increased significantly with the increase in exposure period. However, AChE activity decreased and MNi frequency increased in earthworms after 7 and 14 days of exposure to soils irrigated with TWW for more than a year. The gene expression level of CAT and GST showed a significant variability, thus data are discussed in relation to the studied biomarkers (CAT and GST). These data provide new insights into the effect of toxicity of TWW on the soil's macro fauna, which is strongly affected by the trace elements and other organic compounds accumulated in soils after 20 years of TWW irrigation.