A series of substituted pyrazoline (9a-j) analogues were synthesized via 1,3-dipolar cycloaddition of substituted 2-benzylidene-1-phenylhydrazine (8a-d) with 5-(3-phenylacryloyl)2-hydroxybenzoic acid (5a-f) and the synthesized compounds were characterized by Infrared (IR), Proton Nuclear Magnetic Resonance (1H-NMR), Carbon-13 Nuclear Magnetic Resonance (13C-NMR) and mass spectral techniques. The antimicrobial activity of all the synthesized compounds were screened for both Gram-positive and Gram-negative bacteria along with a fungal strain and the Minimum Inhibitory Concentration (MIC) was calculated. The toxicity of the compounds on human cells were determined by haemolytic activity. The mechanism of action of antimicrobial activity of the synthesized compounds was evaluated by membrane permeabilization assay and confirmed by scanning electron microscopy. Among the synthesized compounds, compounds 9b, 9f and 9g showed strong plasma membrane-permeabilizing activity, further the membrane disruption was confirmed by large dents as observed in E. coli cells under scanning electron microscopy.