With rapid development of novel nanomaterials (NMs), the state of the art technologies with high efficiency and high-throughput characteristics had been applied for nanosafety evaluation. High-content screening (HCS), a cell-based multi-parametric image analysis technique, was adopted in the evaluation of eight different NMs in this study. A set of different endpoints including reactive oxygen species (ROS) production, Ca2+ transient, mitochondrial membrane potential (MMP) and cellular pH levels were checked in human bronchial epithelial (16HBE) cells after incubating with NMs for 24 hours. All NMs induced significant increase of intracellular ROS levels in 16HBE cells, although the decrease of cell viability was only found in Ag and ZnO NMs-treated cells. MMP level had a dose-response decrease in Ag, ZnO and CeO2 NMs-treated cells, while showed a significant increase in TiO2 NMs-treated cells. All tested NMs showed significant up-regulation of cellular lysosomal pH levels. However, none of NMs caused significant changes in cellular Ca2+ level at 24-hour time point. HCS allows for efficient and reliable screening of multiple responses of cells simultaneously within one screen test, which can avoid the problematic interpretation of investigations when carried on a single toxicological endpoint. Therefore, the present data provide insight and inspiration that HCS is an effective and powerful method for image-based assessments with a broad set of biological endpoints in toxicity evaluation of nanomaterials.