The widespread of multi-resistant strains due to the lack of specific treatment and the propagation of infectious diseases requires all resources to remedy this scourge. This study is therefore aimed to assess the antibacterial activity of four synthetic α-Aminophosphonate 4(a-d). Methods: Firstly, α-Aminophosphonate has been synthesized and characterized, then molecular docking of these compounds 4(a-d) into the active binding site of Escherichia coli MurB enzyme (PDB Id: 1MBT) was performed to gain a comprehensive understanding of their biological activity. These compounds have been subjected to in vitro antibacterial screening against three multi-resistant strains E. coli, S. aureus, and L. monocytogenes. These compounds showed crucial antibacterial behavior against all studied strains. Thus, their docking estimation supported the in vitro results and showed that the 4c derivative has considerable binding energy towards the active site of Escherichia coli MurB. These findings provide critical information for the exploration of α-amino phosphonates as novel antibacterial agents.