Pristine TiO2 nanofibers with a range of Ag-loading (wt.% 0, 2, 4, 6 and 8) were prepared by electrospinning technique whose mechanistic insight into their improved antibacterial activity against E.coli as a model microorganism has been investigated. Calcination of as prepared electrospun nanofibers was carried out in ambient air at 500℃ for 3h to promote transformation crystalline phase of TiO2 from anatase to the rutile, removal of reaction moieties and genesis of Ag clusters in these nanofibers. The physicochemical properties of nanofiber samples were analyzed and observed by employing Field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), and Raman spectroscopy. On the whole, these nanofibers have been found to exhibit a smooth surface morphology with minimal beading effect and sustained compositions of the original elements. Heat treatment of the as prepared samples lead to preferential formation of rutile phase as a function of wt.% loading of Ag that strongly supported phase transformation of TiO2 from anatase to rutile. These nanofibers have been tested for their antimicrobial efficacy against the E. Coli bacterium by Well plate method. The results have exhibited formation of consistent zones of inhibition and log10 CFU/mL with the rising content of Ag loading in the TiO2 matrix, while highest antimicrobial efficacy has been observed with the sample containing 8 wt.% of Ag loading. Confocal microscopy and Scanning electron microscopy analysis of treated bacterial samples has disclosed the interaction between nanofibers and bacterial cells, resulting into disruption of cell membrane. This disruption led to the leakage of cell contents and ultimately causing bacterial cell death.