Soils are exposed to nanoparticles (NPs) as a result of their increasing use in many commercial products. Adverse effects of NPs on soil microorganisms have, been reported in several ecotoxicological studies using microcosms. Although repeated exposures are more likely to occur in soils, most of these previous studies were performed as a single exposure to NPs. Contrary to single, contamination, the study of multiple NP contaminations in soils requires the use of specialized setups. Using a soil column experiment, we compared the influence of single and repeated exposures (one, two, or three exposures that resulted in the same final concentration applied) on the transport of titanium dioxide (TiO2) NPs through soil and the effect of these different exposure scenarios on the abundance and activity of soil nitrifying microbial communities after a 2 month incubation. The transport of TiO2 NPs was very limited under both single and repeated exposures and was highest for the lowest concentration injected during the first application. Significant decreases in nitrification-activity and ammonia-oxidizing archaea and bacteria populations were observed only for the repeated exposure scenario (three TiO2 NP contaminations). These results suggest that, under repeated exposures, the transport of TiO2 NPs to deep soil layers and groundwater is limited and that a chronic contamination is more harmful for the soil microbiological functioning than a single exposure.