Nanoparticles (NPs) in biomedical applications have benefits owing to their small size. However, their intricate and sensitive nature makes an evaluation of the adverse effects of NPs on health necessary and challenging. Since there are limitations to conventional toxicological methods and omics analyses provide a more comprehensive molecular profiling of multifactorial biological systems, omics approaches are necessary to evaluate nanotoxicity. Compared to a single omics layer, integrated omics across multiple omics layers provides more sensitive and comprehensive details on NP-induced toxicity based on network integration analysis. As multi-omics data are heterogeneous and massive, computational methods such as machine learning (ML) have been applied for investigating correlation among each omics. This integration of omics and ML approaches will be helpful for analyzing nanotoxicity. To that end, mechanobiology has been applied for evaluating the biophysical changes in NPs by measuring the traction force and rigidity sensing in NP-treated cells using a sub-elastomeric pillar. Therefore, integrated omics approaches are suitable for elucidating mechanobiological effects exerted by NPs. These technologies will be valuable for expanding the safety evaluations of NPs. Here, we review the integration of omics, ML, and mechanobiology for evaluating nanotoxicity.