Ergosterol (ES) and ergosterol peroxide (EP) are secondary metabolites common for different mushrooms and responsible for health promoting effects, including anti-inflammatory, anticancer, antiviral activity, and reduction of the incidence of cardiovascular disease. In this study, a new method for determination of both ES and EP in mushroom extracts was developed. Two methods for UHPLC-MS/MS with the use of APCI and APPI sources were developed and compared. The UHPLC-APPI-MS/MS method was found to be more effective and convenient for the analysis of both compounds in 21 edible and 9 medicinal mushrooms. Among the investigated mushrooms, M. procera was found to contain the highest level of ES, while G. dryophilus constituted the richest source of EP. Therefore, it can be suggested that mushrooms are a valuable source of ES and EP in everyday human diet and can be used for development of nutraceuticals and functional food ingredients.