Escherichia coli Nissle 1917 (EcN) is one of the probiotics that has drawn more attention from researchers in recent days as it extends many host beneficial effects. EcN is being used as a treatment regimen especially for gastrointestinal disorders for more than 100 years. Apart from its clinical applications in its original form, EcN is being genetically engineered to meet the therapeutic requirements which ultimately led to the gradual transformation of EcN from being a mere food supplement to a complex therapeutic agent. However, comprehensive analysis of physiological characterization of EcN is inadequate. In this study, we have systematically studied various physiological parameters and found that EcN grows very well at the normal as well as at stressful conditions such as temperature (30, 37 and 42 °C), nutritional (minimal and LB), pH (ranging from 3 to 7) and osmotic stress (0.4 M NaCl, 0.4 M KCl, 0.4 M Sucrose and salt conditions). However, EcN shows nearly onefold reduction in viability at extreme acidic conditions (pH 3 and 4). It produces biofilm and curlin very efficiently compared to the laboratory strain MG1655. Through genetic analysis we have also shown that EcN exhibits high level of transformation efficiency and greater ability to retain heterogenous plasmid. Very interestingly, we have found that EcN is highly resistant to P1 phage infection. Since, EcN is being exploited largely for its clinical and therapeutic applications, the results that we have reported here would add more value and further expand its scope in clinical and biotechnological research.