Deficiencies in bull mating behaviour have implications for bovine artificial insemination activities. The aim of this study was to identify the compounds present in fluids emitted by cows during oestrus, which could enhance bull libido. Chemical analysis of urine samples from cows led to the characterization of molecules varying specifically at the pre-oestrous and oestrous stages. The synthetic counterpart molecules (1,2-dichloroethylene, squalene, coumarin, 2-butanone, oleic acid) were used to investigate the biological effects on male sexual behaviour and sperm production. When presented to males 2-butanone and oleic acid synthetic molecules significantly lowered mounting reaction time and ejaculation time (-33% and 21% following 2-butanone inhalation, respectively p<0.05). The “squalene + 1,2-dichloroethylene” combination induced a 9 % increase of sperm quantity (p<0.05). This study suggests that the identified oestrous-specific molecules could be part of the chemical signals involved in male and female mating behaviour and may be used for a wide range of applications. The identification of these molecules may have implications for the cattle breeding industry.