Ethnopharmacological relevance Saw palmetto (Serenoa repens) was first used medicinally by native American Indians to treat urological disorders. Nowadays, saw palmetto extracts are widely used in Europe and North America to treat the urinary symptoms associated with benign prostatic hyperplasia even though its mechanisms of action are poorly understood. This study aimed to characterize the bioactive constituents of a lipid extract of saw palmetto that are able to affect contractility of the rat prostate gland. The mechanism of action will also be investigated. Materials and methods A commercially available lipid extract of saw palmetto was subjected to fractionation using normal phase column chromatography. Composition of fractions was assessed by proton nuclear magnetic resonance spectroscopy (1H NMR) and mass spectrometry (MS). Contractile activities of these fractions were evaluated pharmacologically using isolated preparations of rat prostate gland and compared to the activity of the crude extract. Results Saw palmetto extract inhibited contractions of the rat prostate gland which were consistent with smooth muscle relaxant activity. Only the ethyl acetate fraction resulting from chromatography inhibited contractions of isolated rat prostates similarly to the inhibition produced by the crude lipid extract. Comparison with authentic samples and analysis of NMR data revealed that this bioactivity was due to the fatty acid components present in the ethyl acetate fraction. Bioassay using various pharmacological tools identified multiple contractile mechanisms which were affected by the bioactive constituents. Conclusion A fatty acid component of saw palmetto extract causes inhibition of prostatic smooth muscle contractions via a non-specific mechanism.