Actinomycetes are an uncommon agent of human infections and its pathogenic factors are not known. The present study reports a rare case isolation of an actinomycete from a woman pleural fluid; the strain was identified by 16S rRNA gene sequence analysis. This strain was tested to produce an extracellular protease that hydrolysis gelatin, casein and hemoglobin on agar mediums. The purification of the enzyme was carried by ammonium sulfate precipitation, gel filtration and ion exchange chromatographies. The activity of protease was studied at different pH values and temperatures and in the presence of metallic ions and inhibitors. The molecular weight of the enzyme was determined by 12% Tricine SDS-polyacrylamide gel electrophoresis. The strain was identified as Streptomyces cinereoruber ssp. cinereoruber. Extracellular proteolytic enzyme was purified at 19.67 fold and a 3.0% recovery. The enzyme was characterized as having optimal activities at pH 11.0 and 50°C, it keeps more than 50% of activity at pH between 4.0 to 12.0 and it is thermostable at 30 and 40°C. Enzymatic activity is enhanced in the presence of metal ions and inhibited by EDTA and 1,10-phenanthroline. The molecular weight was 53 kDa. This study reports the first case isolation of Streptomyces cinereoruber ssp. cinereoruber from pleural fluid, the extracellular zinc-metalloprotease was proposed as candidate virulence factor. © 2016 Published by CASRP publishing company Ltd. UK. Selection and/or peer-review under responsibility of Center of Advanced Scientific Research and Publications Ltd. UK.