Microorganisms are part of the organic matter in the wastewater. they are present in fecal material and can affect human health. Some chemical and physical agents, such as chlorine, ultraviolet light, reverse osmosis, and silver catalyst are well developed [1,2]. The bactericide activity of silver ions has been known for a long time. The purpose of this study is Preparation and characterization of Ag /4A Zeolite and then investigation of biological application. At first, Ag/4A Zeolite compound was prepared and characterized by using FT-IR, XRD, SEM techniques. The FT-IR data of Ag/4A Zeolite indicate an intense band about ca.1004 cm-1 attributable to the asymmetric stretching of Al–O–Si chain of zeolite. The symmetric stretching and bending frequency bands of Al–O–Si framework of zeolite appear at ca.696 and 466 cm-1, respectively. The XRD pattern of Ag/4A Zeolite showed in Fig.1. The results in Fig. 2 showed that the synthesized Ag/4A Zeolite powder were particle Shape with the diameter of 34-40 nm and were made. Figure 1. The XRD pattern of Ag/4A Zeolite Figure 2. The SEM image of Ag/4A Zeolite Then in vitro antibacterial activity of Ag/4A Zeolite compound was evaluated against Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis), as Gram-positive bacteria, and further against Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), as Gram-negative bacteria. (Table.1) Table.1 The data of biological application Compounds Inhibition zone (mm) Gram-negative bacteria Gram-positive bacteria E. Coli P. aeruginosa S. aureus B. subtilis Ag-A 22 20 20 21 4A Zeolite - - - 30