A Guanidine derivative single crystal has been grown by simple ambient temperature gradient solution growth technique. The as grown single crystals tested against two bacterial pathogens like Bacillus subtilis, Staphylococcus aerus. The result shows that the single crystals have better resistance against the microbes. The standard antiseptic solution like kanamycin, it showed less or equal activity in pathogenic bacteria. In comparison to our currently used Guanidine derivatives single crystals, Guanidine Acetate shows better zone of inhibition against both the pathogens. But in contrast the Guanidine maleate and Guanidine Tetrafluroborate reveals lowest zone of inhibition against Bacillus subtilis and Staphylococcus aerus respectively. It can be concluded that Guanidine acetate exhibit excellent antibacterial effect and therefore, seems to be suitable as a local antiseptic agent, but further clinical studies are necessary.