The negative impact of the synthetic antimicrobials and the overuse of antibiotics have caused global concern, which necessitates the development of a new form of antimicrobials that are derived from natural compounds. In this study, the synthetic route of producing water soluble iso-fatty acid arginate hydrochloride derivatives is investigated by introducing branched (iso-) chain and other linear- (n-) chain fatty acids to the amino acid “arginine” molecules. The two-step synthesis is straightforward and provides an efficient 91%, 88%, and 76% product yield for ethyl n-lauroyl arginate hydrochloride, ethyl n-oleoyl arginate hydrochloride, and ethyl iso-oleoyl arginate hydrochloride, respectively. ATR-FT-IR, NMR, and LC-MS-Q-TOF techniques are used to thoroughly characterize the products. The antimicrobial characteristic of the synthesized ethyl n-lauroyl arginate (LAE) shows a similar activity against both Gram+ and Gram- bacteria as the commercial LAE product. However, its analogs are found to have antimicrobial property primarily against Gram-positive bacteria.