A new series of N-arylamino biquinoline derivatives 5a–x were synthesized under microwave irradiation technique in good yields compared with conventional method and screened for their antimicrobial activity. All the synthesized compounds have been established by elemental analysis, IR, 1 H NMR, 13C NMR and mass spectral data. In vitro antimicrobial activity was carried out against three Gram-positive bacteria (Bacillus subtilis, Clostridium tetani, Streptococcus pneumoniae), three Gram-negative bacteria (Escherichia coli, Salmonella typhi, Vibrio cholerae) and two fungal species (Aspergillus fumigatus, Candida albicans) using broth microdilution method. Of the compounds studied, compound 5u exhibited promising antimicrobial activity against Streptococcus pneumoniae and Salmonella typhi.