We investigated whether TiO2 nanoparticles (NPs) with different size ranges and crystalline forms could affect iron homeostasis in Pseudomonas brassicacearum. In the Pseudomonas species, regulation of iron homeostasis involves two sRNAs known as PrrF. We identified two sRNA-encoding genes prrF1 and prrF2 in the P. brassicacearum genome. To investigate the cytotoxicity of TiO2 NPs, we prepared two shapes of anatase titania particles for physico-chemical characterization before and after in vitro use. We determined their biological activity by analyzing the TiO2 NPs-bacteria interaction using hyperspectral microscopy, as well as their impact on the intracellular iron content. Overall, our results demonstrate that under dark conditions, TiO2 NPs do not have any impact on bacterial growth. We investigated the adaptive response of bacteria and showed that TiO2 NPs induced oxidant stress and consequently altered iron homeostasis. Furthermore, the bacterial iron content significantly decreased, whereas adsorption of TiO2 NPs on bacterial cells increased. Although we used two different crystalline forms that differ in size and shape, they displayed nearly identical mechanisms of toxicity towards bacteria. Finally, we found that the expression of prrF RNA represents a good indicator of intracellular iron status, yielding new insights into the mechanism underlying TiO2 NP toxicity towards bacteria.