This case report describes a two-step protocol for the identification of the causative agent of nocardiosis in a patient with brain abscess, antibiotic susceptibility testing and etiological treatment after neurosurgery. The patient treated with corticosteroids for pulmonary fibrosis and presenting with multiple neurological manifestations was admitted to a neurosurgery clinic. CT and contrast MRI revealed an expansive multilocular lesion 45 x 35 mm in size in the left parietal lobe, differentially diagnosed as malignant glioma. The lesion was biopsied and the histology showed a brain abscess containing white blood cells and dead tissue. The aspirated pus culture yielded bacteria of the genus Nocardia that were further identified, in the first step, by phenotypic methods (Gram positivity, partial acidoresistance, airborne mycelium detection, growth at 45 degrees C, lysozyme resistance and antibiotic resistance phenotype) as belonging to resistance phenotype V., v.s. N. farcinica (resistance to aminoglycosides except amikacin and to third-generation cephalosporins). In the second step of the polyphasic identification, rDNA was isolated and a 1000 bp part of the 16S rRNA gene was sequenced. Sequence comparison with the GenBank database using BLAST software identified the agent as N. farcinica (100%). The isolate was tested for susceptibility by the NCCLS /CLSI dilution method and showed good susceptibility to co-trimoxazole, amikacin and imipenem. The patient was treated with long-term intravenous cotrimoxazole acid in combination with amikacin and his clinical condition and laboratory parameters of inflammation improved. N. farcinica is among the three most frequently isolated Nocardia species in Europe as well as in the Czech Republic where it was repeatedly recovered from the lungs and respiratory tract of immunocompromised patients with systemic nocardiosis.