Introduction The plant family Equisetaceae (Equisetopsida, Monilophyta; common name: horsetails) is part of an ancient group of spore producing plants. Today, Equisetum is the only surviving genus comprising 15 species in two subgenera (Equisetum and Hippochaete). Several unique alkaloids are described to occur in this genus, so far there is very little data on the occurrence and the amount of those alkaloids for the different species. Objective To establish an extraction method and an analytical method to detect and quantify the relevant Equisetum‐type alkaloids and to create a quantitative data set on the alkaloid content of all Equisetum species worldwide. Methodology Hydrophilic interaction liquid chromatography high‐performance liquid chromatography tandem mass spectrometry (HILIC HPLC‐MS/MS) in electrospray ionisation (ESI) positive mode was used to analyse and quantify the alkaloid content of 68 Equisetum samples. Results The presence of nicotine in at least one sample of each Equisetum species could be demonstrated. The total nicotine amount rarely exceeded 250 μg/kg and 50 μg/kg for the subgenus Equisetum and Hippochaete, respectively. Besides nicotine, Equisetum‐type alkaloids (mainly palustrine and palustridiene) were only detected in three species, namely E. palustre, E. bogotense and E. giganteum. For E. giganteum, palustridiene was detected at levels around the limit od detection (LoD) (25 μg/kg), whereas in E. palustre and E. bogotense, both alkaloids (palustrine and palustridiene) were detected at much higher levels (20–800 mg/kg). Conclusions All Equisetum species occurring worldwide were successfully subjected to a detailed qualitative and quantitative alkaloid analysis using a newly developed HILIC‐HPLC‐ESIpos‐MS/MS approach. The data set can be used to distinguish different Equisetum‐chemotypes.