Hydraulic fracturing fluids are chemical mixtures used to enhance oil and gas extraction. There are concerns that fracturing fluids are hazardous and that their release into the environment - by direct injection to coal and shale formations or as residue in produced water - may have effects on ecosystems, water quality and public health. This study aimed to characterise the acute cytotoxicity of a hydraulic fracturing fluid using a human gastrointestinal cell line and, using this data, contribute to the understanding of potential human health risks posed by coal seam gas (CSG) extraction in Queensland, Australia. Previous published research on the health effects of hydraulic fracturing fluids has been limited to desktop studies of individual chemicals. As such, this study is one of the first attempts to characterise the toxicity of a hydraulic fracturing mixture using laboratory methods. The fracturing fluid was determined to be cytotoxic, with half maximal inhibitory concentrations (IC50) values across mixture variations ranging between 25 and 51 mM. When used by industry, these fracturing fluids would be at concentrations of over 200 mM before injection into the coal seam. A 5-fold dilution would be sufficient to reduce the toxicity of the fluids to below the detection limit of the assay. It is unlikely that human exposure would occur at these high ('before use') concentrations and likely that the fluids would be diluted during use. Thus, it can be inferred that the level of acute risk to human health associated with the use of these fracturing fluids is low. However, a thorough exposure assessment and additional chronic and targeted toxicity assessments are required to conclusively determine human health risks.