Microorganisms that grow in the environment may enter the drinking-water and attach to and grow on drinking-water pipes and other surfaces, forming biofilms as the most common means of growth in the environment and once established, biofilms are difficult to eradicate. Antimircobial agents, mainly chlorine, are generally used to disinfect drinking water, however. There are not yet standardized disinfection strategies with reliable efficacy and the development of novel anti-biofilm strategies is still of major concern. In the present study bacterial diversity and the prevalence of Shiga Toxin-producing E.coli (STEC) were detected in Drinking Water Distribution System (DWDS) in Lebanon. Bacterial isolates belonging to coliform group were revealed as the most prevalent isolates. Different E.coli isolates under test (7 isolates) revealed as different strains showing different susceptibilities to commonly used antibiotics were then tested for the presence of stx1, stx2, eaeA, ehlyA and fliC virulence genes using PCR. The seven E.coli isolates were considered as STEC serotypes, carrying virulence marker genes. However, none of these isolates were positive for the fliC gene and were thus H7 negative, and could not be classified as O157:H7. Biofilm production of all the isolates under test was quantitatively investigated using the method of adherence to polystyrene mirotiter plates, where 32.26% of the isolates were detected as strong, 51.61% as moderate and 16.13% as weak biofilm producers. Pre-coating microtiter plates with tap water showed a significant increase in biofilm production in all the tested isolates. Treatment with cell-free supernatant (CFS) of Lactobacillus acidophilus and Streptomyces sp. one at a time at 20% concentration resulted in average biofilm inhibition (52.89 and 39.66% respectively) with no bacteriostatic or bactericidal activity against all the tested isolates. Moreover, pre-coating wells with supernatants revealed that Lactobacillus acidophilus CFS inhibited average biofilm formation (62.53%) by altering the adhesion of bacterial isolates to the surface, preventing the initial attachment step, which is important for biofilm production. However, Streptomyces sp. CFS was able to inhibit biofilm formation using another strategy which might interfere with the bacterial Quorum Sensing (QS) system, where most of the isolates under test were shown to produce QS signaling.