Titanium dioxide (TiO<inf>2</inf>) nanoparticles (NPs) are commonly used materials present in many consumables for which most people are exposed to. The biological hazards of the NPs on human health have been demonstrated previously. In this study, we aimed to assess the cytotoxicity potency of TiO<inf>2</inf> NPs on the primary human amniotic fluid cells. The cells derived from amniotic fluid were treated with different dosages of TiO<inf>2</inf> NPs for some periods. Cell adhesion status was assessed using a light microscopic observation. Cell proliferation and cell death rates were determined using trypan blue staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Also, mitotic index was determined using fluorescence in situ hybridization with chromosome 8 centromer-specific DNA probe. Disrupted cell adhesion, decreased proliferation, and increased mortality rates were detected in the cells that were treated with TiO<inf>2</inf> NPs depending on the dosage (p < 0.001). Also, reduced mitotic index was determined in the cells depending on the time and TiO<inf>2</inf> dosage when compared with the controls (p < 0.0001). These results showed that TiO<inf>2</inf> NPs have high cytotoxicity for amniotic fluid-derived cells. Therefore, different products containing TiO<inf>2</inf> NPs should be used with care, especially for pregnant women.