Jordan is one of the lowest countries in the world in terms of water resources. The reuse of treated wastewater is an important alternative to supply agricultural demands for water. In Jordan, Kherbet Al-Samra wastewater treatment plant (KSWWTP) is the largest and its effluent is mainly used for irrigation purposes. In this study, bacterial contamination and mutagenic potential were evaluated in six sites, beginning with KSWWTP and ending with King Tallal Dam. The results showed high contamination with many pathogenic bacteria and coliforms. The isolated pathogenic bacteria were Salmonella sp., Shigella sp., Bacillus cereus and Staphylococcus aureus. The isolated opportunistic pathogenic bacteria were Acinetobacter lwoffii, Elizabethkingia meningosepticum, Pseudomonas fluorescens and Bacillus licheniformis. These bacteria were found in all sampling sites without a specific prevalence pattern. Differences in temperature between seasons affect total coliform and other bacterial count. All water samples showed positive mutagenic activity and high bacterial pollution. Improving the disinfection efficiency in the wastewater treatment plant is important to minimize potential toxicity and exposure of public health to pathogenic bacteria, reduce water resources' contamination and environmental pollution. Increasing effluent sampling frequency from KSWWTP is required to monitor bacterial contamination and toxicity/mutagenicity level for water safety and public health risk assessments.