The nutritional properties of Pleurotus mushrooms were studied to select the varieties with the most favourable properties. These mushrooms have high nutritional value; they are rich in carbohydrates, protein, minerals, vitamins, chitin and reducing compounds, such as phenols and polyphenols. In this study, the polyphenol profiles of thirteen Pleurotus ostreatus cultivars were established by the UHPLC-ESI-MS/MS technique. The results showed that 4-hydroxibenzoic acid, caffeic acid, p-coumaric acid and vanillic acid were the most abundant polyphenolic components in the samples. In addition, the Fourier-transformed near infrared (FT-NIR) spectra of the samples were recorded and evaluated. The correlation between the differences in NIR spectra and the differences in polyphenol patterns of the samples was investigated. The polyphenol results were subjected to several statistical evaluations (Kruskal–Wallis test, Principal Component Analysis (PCA), Spearman correlation analysis, cluster analysis) to detect possible differences between the samples. Relationships between the polyphenol profile and antioxidant capacity (FRAP), total polyphenol content (TPC), free amino acid content (fAA) and the values of each polyphenol component were examined. Based on the results, an effort was made to group the varieties according to the attributes tested.