Background: The spread of antibiotic resistance has become one of the most prominent health challenges in recent years especially in water. Shatt Al-Diwaniyah (Al-Diwaniyah river) is the primary source of drinking and irrigation water in Al-Diwaniyah Province. The current study aims to evaluate the percentage of contamination of this waterfall with coliform bacteria and determine whether it carries antibiotic resistance or not. Method: Our study included collecting 60 water samples from different areas along the Shatt Al-Diwaniyah. The bacteria were investigated using culture media and biochemical tests, while the resistance gene (integron class 1) was investigated using the polymerase chain reaction (PCR) method.  Antibiotics sensitivity test was performed against ten different antibiotics by disc diffusion method. Results: The results showed that Escherichia coli (E. coli) isolates were contaminants of all water samples, while we found Enterobacteria and Serratia in 70% and 3% respectively of the samples. Most of the isolates appeared resistant to penicillin, ampicillin, amoxicillin, and tetracycline. The integron class 1 was identified in E. coli and Enterobacter isolates at a rate of 82% and 55% respectively. The isolates carrying this gene were multi-resistant to antibiotics. Conclusion: We found E. coli and Enterobacter to be the main contaminated coliform of Shatt Al-Diwaniyah water, and most of them were multi-resistant to antibiotics and carried integron class 1.