Rhodococcus (R.) equi is a pathogen primarily known for infections in equine foals, but is also present in numerous livestock species including New World camelids. Moreover, R. equi is considered an emerging zoonotic pathogen. In this report, we describe in detail a fatal rhodococcal infection in an alpaca (Vicugna pacos), to our best knowledge, for the first time. The alpaca died due to a septicemic course of an R. equi infection resulting in emaciation and severe lesions including pyogranulomas in the lungs and pericardial effusion. The onset of the infection was presumably caused by aspiration pneumonia. R. equi could be isolated from the pyogranulomas in the lung and unequivocally identified by MALDI-TOF MS analysis and partial sequencing of the 16S rRNA gene, the 16S-23S internal transcribed spacer (ITS) region and the rpoB gene. The isolate proved to possess the vapA gene in accordance with tested isolates originating from the lungs of infected horses. The R. equi isolates revealed low minimal inhibitory concentrations (MIC values) for doxycycline, erythromycin, gentamycin, neomycin, rifampicin, trimethoprim/sulfamethoxazole, tetracycline and vancomycin in antibiotic susceptibility testing. Investigations on the cause of bacterial, especially fatal, septicemic infections in alpacas are essential for adequately addressing the requirements for health and welfare issues of this New World camelid species. Furthermore, the zoonotic potential of R. equi has to be considered with regard to the One Health approach.