This study focuses on the health benefits of several fresh herbs that are commonly used in the Mediterranean diet. Antioxidant activity, phytosterol content and α-amylase inhibitory activity of fresh basil, lavender, oregano, rosemary, sage, and thyme are analyzed and compared. High-performance thin-layer chromatography (HPTLC) combined with effect directed analysis was used to detect and quantify biological active compounds on chromatograms. The highest antioxidant activity was measured in the extract from oregano leaf, while the highest terpenoid content was in basil leaf extract. All extracts except lavender leaf and lavender flower extracts showed α-amylase inhibition. The same compound at hRF = 68 in basil, oregano, rosemary, sage, and thyme extracts was responsible for α-amylase inhibition. Combined with effect-directed assays and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, hyphenated HPTLC allowed a fast characterization of the active compound. ATR spectral analysis of this band tentatively identified oleanolic acid (or its derivative) to be responsible for the α-amylase inhibition.