The antimicrobial activity of seven α-aminophosphonate derivatives was evaluated. These new compounds were synthesized via a one-pot reaction of the Kabachnik-Fields between 2-((1H-benzo[d]imidazol-2-yl)thio)acetohydrazide, aldehyde derivatives, and diethyl phosphite in the presence of magnesium triflate as a catalyst. The novel compounds are identified by analytical and spectroscopic techniques. The use of magnesium triflate as a catalyst revealed short reaction time (1 h), in 10 mol%.The activity of the new compounds was against the Gram-positive bacteria Bacillus cereus, and Staphylococcus aureus, as well as the Gram-negative bacteria Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginose, and the fungus Candida albicans. The results indicated that most of the new compounds have moderate to high potency as antibacterial and antifungal agents. And the theoretical study confirmed that the compounds excel in binding with a more stable binding energy than the antibiotic penicillin, and this was confirmed by laboratory results. In the case of Escherichia coli ATCC® 25922™, only 5a and 5e displayed significant inhibitory activities equal to or greater than the reference drug. Most of the new compounds (5c–5 g) revealed high potency against Salmonella typhimurium ATCC® 14028™. The highest value is for 5d which has the high inhibitory activity against Cadida albicans.