Three N-glycoconjugates have been condensed with four aromatic aldehydes to afford imine linkage containing twelve new compounds. These compounds were tested for antibacterial activity against three bacteria [gram positive (Bacillus cereus) and gram negative (Escherichia coli and Klebsiella pneumoniae)] and antifungal activity against Fusarium graminearum, Fusarium monilliforme and Aspergillus flavus. All the compounds exhibited fair amount of antibacterial activities against both types of bacteria, but antifungal activity was exhibited by only three compounds, which contained naphthyl moiety. Microbial cell penetration ability of two most efficient compounds and discrimination of live/dead cells have been explored using microscopic technique. Effect of these two compounds on bacterial nucleic acids have also been studied, which helped in understanding the mechanistic insights of cell death.