The inadequate treatments given to the served waste water which are disposal to the rivers and sea coast are the major sources of faecal Microorganisms and enteric bacterial pathogens. They are among the most serious effects of water pollution bringing risks on public health. None of the current methods for detection of pathogens offer real-time on site solutions, are capable of delivering a simple visual detection signal, or can be easily instrumented as an indicator of the presence of a pathogen in water. The use of lipid vesicles incorporating Polydiacetylenes (PDAs) for the development of biosensors for “real-time” detection of pathogens has become an alternative, due to its potential for simple colorimetric response against harmful environmental effectors. However, its actual application in the field has been complicated because lipid vesicles are unable to respond specifically to environmental changes. In this paper, we report several experimental trials leading to improved response in the detection of flagellated pathogens in drinking water. Chromatic biomimetic membranes of TRCDA/DMPC and TRCDA/DMPC/Tryptophan were used in agar and liquid media, which were challenged with different amounts of Escherichia coli and Salmonella typhimurium. In addition, the effect of some divalent cations on the interaction with vesicles TRCDA/DMPC was investigated. The results indicated an improvement in the response times, both visually and quantitatively, through the use of TRIS-EDTA and proper growing conditions for E. coli and Salmonella. With the application of both conditions, it was possible by incubation at 35ºC to promote bacterial growth, therefore avoiding a dramatic effect on the colour change over control samples which may invalidate the test. Our experiments indicated that the minimum bacterial concentration necessary to produce the transition from blue to red on the vesicles as biosensor approaches 10(8) CFU/ml within 4 hrs, faster than traditional methods such as MPN or plate count agar. We present here incubations of samples of contaminated water at 35ºC, in agar plates containing chromatic biomimetic membranes of TRCDA/DMPC. A measurable colour transition is obtained within a reaction time of four hrs, which compares favourably with detection times between seven to 24 hrs corresponding to available tests.