The most of secreted proteins are exported by Sec translocase (Secretion pathway). SecA ATPase is the preprotein translocase nanomotor that undergoes membrane insertion and deinsertion to drive preprotein across the bacterial inner membrane, which is unique and indispensable to bacteria. It should be presumed that the compound which inhibits the activity of SecA ATPase probably can be used as the candidate of bactericide. Pseudomonas aeruginosa secA gene product, whose amino acid sequence displays PaSecAN75 (The N-terminal first 645 amino acids of Pseudomonas aeruginosa SecA), was cloned, overexpressed and purified in order to establish enzyme inhibitor screening model. After establishment and optimization of screening assay, a primary screening was performed with compounds library and microbial fermentation collection in our institute. 4 out of 3220 compouds and 66 out of 7196 microbial fermentation samples were identified as primary hit. To confirm the hits from the primary screening, a cell-based SecA activity assay was used to assess their inhibitory effect in vivo. Results showed that 3 compounds and 6 microbial fermentation samples inhibited enzymic activity of SecA in vitro and impaired its function in vivo.