OBJECTIVE: Molecular determinants of β-lactam resistance are poorly explored for most Nocardia species, such as Nocardia brasiliensis. In this study, we characterized resistance mediated by two β-lactamases in the reference strain N. brasiliensis HUJEG-1 and extended our analysis to nine N. brasiliensis clinical strains. METHODS: The susceptibility of N. brasiliensis HUJEG-1 was determined by measuring the MIC by microdilution for five β-lactam antibiotics associated or not with β-lactamase inhibitors (clavulanate and avibactam, 4µg/mL). Two putative class A β-lactamase encoding genes (bla RESULTS: N. brasiliensis HUJEG-1 was resistant to amoxicillin, cefuroxime and cefotaxime, but susceptible to their combination with clavulanate or avibactam. This strain was resistant to imipenem (with or without inhibitors) and susceptible to meropenem. BRA-1 showed high catalytic efficiencies against penams and cephems, but not against penems, suggesting that imipenem resistance was mediated by another mechanism. The hydrolytic activity of BRS-1 was 100- to 1000-fold lower than that of BRA-1 for all β-lactams tested, suggesting that BRS-1 has a minor contribution to β-lactam resistance. Analysis of the nine clinical strains showed variations in susceptibility to cefotaxime, as well as diversity in genetic backgrounds and BRA-1 sequences. CONCLUSION: N. brasiliensis HUJEG-1 resistance to penams and cephems was mainly due to the class A β-lactamase BRA-1.