Two kinds of nanoscale TiO2 films were prepared by magnetron sputtering and screen printing methods, respectively. Results show that both phase composition and specific surface area of the film affect the photocatalytic bactericidal efficiency. Time-series in situ atomic force microscopy (AFM) observation were further used to characterize the cellular responses of Escherichia coli (E. coli) in photocatalytic process. Some nanosized patches were found on the bacterial surface in the forepart of photocatalytic reaction. It suggested that the photocatalytic attack induced the self-protection of bacteria at first. Subsequently, some cracks on the surface and the enlargement of cell body indicated that the cell wall was damaged and lost its structure supporting function, and it eventually led to the death of bacteria.