Teleost fishes use olfaction to detect natural chemical compounds – odorants - belonging to several different classes. These odorants can be released by conspecifics or other species enabling fish to navigate, locate food, identify predators, and recognize conspecifics. The Lusitanian toadfish (Halobatrachus didactylus) is a bottom-living species that uses acoustic communication during the reproduction season; reproductive males vocalize to attract females to their nest and take care of the nest and the eggs, after reproduction. As other teleosts, the toadfish has a well-developed olfactory system. Recent data showed that the toadfish has high olfactory sensitivity (as assessed by the electro-olfactogram - EOG) to a range of amino acids and intraspecific body fluids, particularly intestinal and accessory glands fluids, with quantitative differences between those from reproductive and non-reproductive males. In this study, we focused on the intestinal fluid (IF) from males. Using solid-phase extraction and preparative HPLC, we fractionated the IF collected from reproductive and non-reproductive males; 31 fractions from each were obtained and their olfactory activity was assessed by EOG. Cross-adaptation experiments with bile acids as adapting solutions indicated that the olfactory-active fractions likely contain bile acids or share olfactory receptors with them; however, those from reproductive males are qualitatively different from those from non-reproductive males. This suggests a role for these odorants – likely bile acids – in reproduction.