With the emergence of multi-drug resistant bacteria and hospital-acquired infections, there is an urgent need to develop new antibiotics. Here, we report the synthesis, physico-chemical characterizations, and antimicrobial activity assays of four Azo compounds that differ in their alkyl chain length. The molecular mechanism of their antibacterial activity was investigated by complementary in vitro and in silico biophysical studies. The compounds with alkyl chain lengths of four or six carbons showed a low MIC 50 against Escherichia coli and Bacillus subtilis. Our investigations into the mechanism of their action revealed that phosphatidylethanolamine in the bacterial plasma membrane plays an important role in their antibacterial activity.

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