The misuse of conventional antibiotics and natural selection of the infectious bacterial population has produced drug resistance. Thus, novel effective antibiotic compounds that treat bacterial infections resistant to available therapies are needed. SecA is an indispensable ATPase of the protein translocation machinery present in all bacteria. SecA is responsible for the secretion of many essential proteins, some toxins and virulence factors, and is essential for bacterial survival. SecA has no counterpart in mammalian cells, thus provides an ideal target for developing antimicrobial agents. SCA-13 (HO) is a pyrimidine analog derived from virtual screening; it exerts the ability to inhibit SecA translocation ATPase activity with an IC50 of 75 µM. HO showed promising bacteriostatic activities against a vacomycin resistant strain of S. aureus Mu50 and B. anthracis Sterne. No significant difference in antimicrobial activity of HO was observed among efflux pump strains of S. aureus, suggesting that compound HO is not a substrate of NorA or MepA efflux pumps. Resistant mutants of E. coli NR698 selected from HO need to be characterized to gain a better understanding of the resistance mechanisms and subsequently will allow for the identification of the drug target.