The current study is conducted to detect bacterial contamination of groundwater in Al-Dour district within Salah al-Din Governorate for a distance extending about 35 km by eight wells with depths ranging between (65-90) meters during the study period extending from spring 2018 to winter 2019. The study includes isolation and identification of the most important species of bacteria contaminating well water and investigating its virulence factors with molecular diagnosis. The Isolates were detected as being able to produce some virulence factors, which included hemolycin, lipase and protease, lecithinase, biofilm, urease, and beta-lactamases, is detected. The results show that Klebsiella pneumoniae produces biofilm, hemolysin, lipase and lecithinase by 100%, while beta-lactamase and both urease and protease enzymes are not produced. As for Enterobacter cloacae, it produced hemolysin, protease and lipase by 100%, while it produced the enzymes beta-lactamase and lecithinase by 50%, and did not form or produce the biofilm and the urease enzyme. Concerning Aeromonas veronii, it produced beta-lactamase, lipase, biofilm, protease and lecithinase by 100% and hemolysin by 50% and did not produce urease enzyme. Enterococcous cecorum produced beta-lactamase, urease, protease and biofilm by 100%, and produced hemolysin and lipase enzymes by 66.7% and lecithinase enzyme by 33.3%, while Enterococcous columbae produced beta- lactamases, urease and biofilm by 100% but unable to produce lipase, lecithinase, hemolysin and protease. Granulicatella elegans were shown to be producing beta-lactamase, lecithinase, biofilm and protease by 100%, but did not produce hemolysin, urease and lipase enzymes. As for Aeromonas hydrophila, they appeared to be producing lipase, urease and hemolysin by 100%, but produced protease, lecithinase and beta-lactamase by 50% but did not produce the biofilm. The results of Polymerase Chain Reaction (PCR) show the use of specialized primers of some genes of virulence factors in the isolated bacteria. The protease gene encoded ProA and beta-lactamases gene encoded MOXM are found in Enterobacter cloacae, the hemolysin gene encoded HemK and the biofilm gene encoded LuxS found in Klebsiella pneumonia, the urease gene encoded UreA and lipase gene encoded lip found in Aeromonas hydrophila. As for A.veronii, it has been detected holding the protease gene encoded ProA and the lipase gene encoded lip the. With respect to the genera Enterococcus sp., Tuf biofilm encoded is found in both Enterococcous columbae and Enterococcous cecorum. In Granulicatella elegans, the presence of the RpoB gene is detected.

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