Preharvest calcium application has been shown to increase broccoli microgreen yield and extend shelf life. In this study, we investigated the effect of calcium application on its metabolome using ultra-high-performance liquid chromatography with mass spectrometry. The data collected were analyzed using principal component analysis and orthogonal projection to latent structural discriminate analysis. Chemical composition comparison shows that glucosinolates, a very important group of phytochemicals, are the major compounds enhanced by preharvest treatment with 10 mM calcium chloride (CaCl2). Aliphatic glucosinolates (glucoerucin, glucoiberin, glucoiberverin, glucoraphanin, pentyl glucosinolate, and hexyl glucosinolate) and indolic glucosinolates (glucobrassicin, neoglucobrassicin, and 4-hydroxyglucobrassicin) were increased significantly in the CaCl2 treated microgreens using metabolomic approaches. Targeted glucosinolate analysis using the ISO 9167-1 method was further employed to confirm the findings. Results indicate that glucosinolates can be considered as a class of compounds that are responsible for the difference between two groups and a higher glucosinolate level was found in CaCl2 treated groups at each time point after harvest in comparison with the control group.