Environmental resistance is an important factor for understanding the epidemiology of leptospirosis. Recently, new Leptospira hosts were identified, including also marine mammals. Moreover, halotolerant Leptospira strain, isolated from the environment and animals, highlighted the capability of this microorganism to persist in the seawater. The aim of this research was to investigate the bacteriostatic and bactericidal effect of salt on Leptospira strains belonging to 16 different serovars. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were verified through the microdilutions method starting from a 20% sodium chloride concentration. MIC values obtained were between 0.3125% and 10% of salt, while MBC values between 0.625% and >20%. Icterohaemorrhagiae (MIC: 0.3125%; MBC: 0.625%) resulted the most inhibited serovar, while the most resistant was Tarassovi (MIC: 10%; MBC: >20%). Interestingly, trends were reported for Pomona (MIC: 1.25%; MBC: >20%) and Bratislava (MIC: 0.625%; MBC: 20%), highlighting low MIC values but high MBC values. This is the first investigation aimed at the in vitro effect of salt on the growth of Leptospira spp. reference strains.