A suspect screening LC-HRMS workflow for polyphenols was successfully applied to seven mushroom and two plant species known to contain polyphenol oxidases. This allowed for the comprehensive identification and annotation of polyphenols present in this limited sample set. Moreover, (semi-)quantification was performed on conjugated and unconjugated polyphenols from 13 the untargeted data, and the samples were additionally quantified with a targeted LC-MS/MS assay covering a wide range of polyphenol standards. Overall, a similar number of flavonoids and nonflavonoids were detected, with roughly two-thirds of the flavonoids conjugated to a sugar moiety. Tickseed flower petals showed a high number and variety of polyphenols, among which isomers of hesperetin-O-glucoside predominated. The mushroom species exhibited similar polyphenol profiles, especially the two button mushrooms. These results yielded insights into the polyphenols naturally present in these species. However, a better overview should be obtained in the future, especially taking into account the influence of confounding factors, such as growing conditions, and an increased number of biological replicates since in this preliminary work only one sample per species was included. Moreover, a fully validated LC-MS/MS method should be utilized, to allow for more accurate quantification. Lastly, the comprehensive profiling of these species is intended to aid in defining and interpreting the selectivity of PPOs in the future, by, for example, investigating interactions between specific polyphenols detected and isolated PPO isoenzymes.