Chemotaxis allows bacterial cells to migrate towards or away from chemical compounds. In the present study, we developed a microscopic agar-drop assay (MAA) to investigate the chemotactic behaviour of a coiled spirochete, Leptospira biflexa. An agar drop containing a putative attractant or repellent was placed around the centre of a flow chamber and the behaviour of free-swimming cells was analysed under a microscope. MAA showed that L. biflexa cells gradually accumulated around an agar drop that contained an attractant such as glucose. Leptospira cells often spin without migration by transformation of their cell body. The frequency at which cells showed no net displacement decreased with a higher glucose concentration, suggesting that sensing an attractive chemical allows these cells to swim more smoothly. Investigation of the chemotactic behaviour of these cells in response to different types of sugars showed that fructose and mannitol induced negative chemotactic responses, whereas xylose and lactose were non-chemotactic for L. biflexa. The MAA developed in this study can be used to investigate other chemoattractants and repellents.