Two novel microwave-assisted extraction (MAE) methods were developed for the isolation of phenols and tocopherols from pistachio nuts. The extracts were analyzed by reversed-phase high-pressure liquid chromatography coupled with a UV detector (RP-HPLC-UV). In total, eighteen pistachio samples, originating from Greece and Turkey, were analyzed and thirteen phenolic compounds, as well as α-tocopherol, (β + γ)-tocopherol, and δ-tocopherol, were identified. The analytical methods were validated and presented good linearity (r2 > 0.990) and a high recovery rate over the range of 82.4 to 95.3% for phenols, and 93.1 to 96.4% for tocopherols. Repeatablility was calculated over the range 1.8–5.8%RSD for intra-day experiments, and reproducibility over the range 3.2–9.4%RSD for inter-day experiments, respectively. Principal component analysis (PCA) was employed to analyze the differences between the concentrations of the bioactive compounds with respect to geographical origin, while agglomerative hierarchical clustering (AHC) was used to cluster the samples based on their similarity and according to the geographical origin.