Groundwater aquifers are the largest source of drinking water for human population. Current available information of the quality of groundwater is quite limited mainly due to the lack of comprehensive analysis of groundwater and the challenging task of applying any analytical method in its investigation. In this thesis, a new method based on “soft” mass spectrometry and differential ion mobility (FAIMS) was developed to discover previously unknown sulphur-containing contaminants in groundwater in Ontario. Following this discovery, de novo identification of these contaminants was accomplished by determining their elemental composition based on mass measurements and their chemical structures from unique dissociation patterns. The compounds characterized in this study were found to be thiotetronic acids which are structurally related to synthetic and natural antibacterial agents such as the natural antibiotics thiolactomycin and thiotetramycin, allowing for speculation as to their potential beneficial properties.