A novel fast-setting calcium silicate cement with fluoride (CSC) has been developed for potential application in tooth crowns. This study compared the cytotoxicity of CSC compositions and a variety of dental materials. We tested CSC compositions (Protooth), MTA, Biodentine, Ketac Molar, Fuji II LC, Vitrebond, DeTrey Zinc, Dycal, and IRM, DMEM (negative control) and 1% NaOCl (positive control). After setting of cements for 24 h, specimens were immersed in DMEM for 24 h to obtain material elutes. The elutes were serially diluted in serum-free DMEM to obtain three dilutions. L929 mouse fibroblast cells (1 × 10 cells per well) were treated for 24 h with elute dilutions (n = 3). Cytotoxicity was determined using methyl-thiazolyl-tetrazolium assay in triplicate. CSC compositions, MTA, and Biodentine showed no significant reduction in cell viability compared to DMEM. There was no significant difference in cell viability, at any of three dilutions, between CSC compositions and either MTA or Biodentine. Cytotoxicity was significantly lower for CSC compositions than for Vitrebond, DeTrey Zinc, Dycal, IRM, and 1% NaOCl, at all three dilutions, and undiluted Fuji II LC elute. In contrast to resin-modified glass ionomers, zinc phosphate cements, Dycal, and IRM, the CSC compositions showed no cytotoxic potential.