Subtropical soil microbial isolates were screened for carbohydrate, tributyrin, or olive oil hydrolysis using agar plates supplemented with the corresponding substrates. A heterotrophic, aerobic, Gram-positive strain displaying activity on tributyrin was selected and further characterized. Analysis of the morphological and physiological traits of the strain placed it as a member of the genus Rhodococcus. Further 16S rDNA sequencing revealed a 99% identity to Rhodococcus erythropolis. The strain displayed lipolytic activity on fatty-acid-derivative substrates of short chain length, with cell extract fractions having highest activity, as confirmed by the presence, after zymogram analysis, of a ca. 60-kDa intracellular protein band with activity on 4-methylumbelliferone-butyrate substrate. The presence of such a lipolytic enzyme, similar to those found in other Gram-positive bacteria, indicates that the strain could be of interest for certain biotechnological applications, like the synthesis of pharmaceuticals or biocide detoxification.