The identification and antimicrobial susceptibility of Nocardia spp. are essential for guiding antibiotic treatment. We investigated the species distribution and evaluated the antimicrobial susceptibility of Nocardia species collected in southern Taiwan from 2012 to 2020. A total of 77 Nocardia isolates were collected and identified to the species level using multi-locus sequence analysis (MLSA). The susceptibilities to 15 antibiotics for Nocardia isolates were determined by the broth microdilution method, and the MIC50 and MIC90 for each antibiotic against different species were analyzed. N. cyriacigeorgica was the leading isolate, accounting for 32.5% of all Nocardia isolates, and the prevalence of Nocardia isolates decreased in summer. All of the isolates were susceptible to trimethoprim/sulfamethoxazole, amikacin, and linezolid, whereas 90.9% were non-susceptible to cefepime and imipenem. The phylogenic tree by MLSA showed that the similarity between N. beijingensis and N. asiatica was as high as 99%, 73% between N. niigatensis and N. crassostreae, and 86% between N. cerradoensis and N. cyriacigeorgica. While trimethoprim/sulfamethoxazole, amikacin, and linezolid remained fully active against all of the Nocardia isolates tested, 90.9% of the isolates were non-susceptible to cefepime and imipenem.