In conclusion, we identified a new esterase EstAS belonging to family III lipases from SBR activated sludge metagenomic library. EstAS is a very interesting enzyme with high potential for downstream biotechnological applications. This was confirmed by extensive biochemical characterization, substrate specificity, stability towards addictives including metal ions and detergents, and also, wide pH and temperature spectra. This study also demonstrated that the metagenomic approach is very useful for expanding our knowledge of enzyme diversity, especially for bacterial esterases. Accessing the metagenomic pool of lipases and esterases can be an immediate source of novel biocatalysts, or yield enzymes that can be further specialized by directed evolution.