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Western people now spend close to 90% of their time indoors, one-quarter of which occurs at their place of employment. As such, interactions between employees and the workplace built environment have significant potential impact on employee health and safety. However, the range of workers’ daily chemical exposures is still poorly understood. Likewise, the influence of workers themselves and of worker behavior on the chemical composition of the workplace is still unknown. In this case study, we used untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare the chemical signatures of three different types of workplaces: scientific research buildings, office buildings, and one mixed-purpose building. Our results identified differential signatures of public building surfaces based on building purpose, sampling location and surface materials. Overall, these results are helping define the influence of human behavior on the workplace chemical environment and identify the chemical hazards to which people are exposed throughout their workday.HighlightsImplementation of untargeted liquid chromatography-tandem mass spectrometry to study workplace chemical exposures.Shared chemical signatures were identified based on building purpose.Differential chemical signatures were identified based on surface material and sampling location.Annotated molecules include pharmaceuticals, illicit drugs, food chemicals, constituents of paints and stains, and cleaning products.