Leptospirosis is a worldwide zoonotic disease determined by pathogenic spirochetes of the genus Leptospira. The control of bovine leptospirosis involves several measures including antibiotic treatment of carriers. Despite its importance, few studies regarding antimicrobial susceptibility of strains from bovine origin have been conducted. The aim of this study was to determine the in vitro susceptibility of Leptospira strains obtained from cattle in Rio de Janeiro, Brazil, against the main antibiotics used in bovine veterinary practice. A total of 23 Leptospira spp. strains were investigated for minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) using broth macrodilution. At the species level, there were not differences in MIC susceptibility except for tetracycline (P < 0.05). Nevertheless, at the serogroup level, differences in MIC were observed among Sejroe strains, mainly for ceftiofur, doxycycline and in MBC for streptomycin (P < 0.05). One strain presented MBC values above maximum plasmatic concentration described for streptomycin and was classified as presenting reduced susceptibility. Efficacy of antimicrobial therapy on bovine leptospirosis could be compromised due to occurrence of infection by Leptospira strains presenting reduced susceptibility.