Antimicrobial resistance reduces the effectiveness of the standard treatment and also increases the risk of spreading the disease to others. Therefore, there is an urgent need for the development of new class of antimicrobials. In such an attempt, benzothiazole schiff base hybrids synthesised were evaluated for antimicrobial potency against E. coli, S. typhi, S. aureus, E. faecalis, K. pneumonia, P. aeruginosa, C. albicans, C. tropicalis, and C. krusei and also an attempt was made to understand their molecular mechanism of action. Some of the benzothiazole schiff base hybrids have exhibited excellent antimicrobial activity with MIC of 3.91µg/ml against S.aureus, P.aeruginosa and E.coli in comparison with the standard drug ciprofloxacin. The molecular mechanism of action of benzothiazole schiff base hybrids was elucidated in both intact bacterial cells and plasmid DNA. The potent antimicrobial activity of the hybrids may be due to membrane perturbing mode of action explicated by membrane depolarization assay and fluorescent assisted cell cytometry (FACS) study on intact bacterial cell. However, the activity may also be due to the intracellular mode of action explicated by DNA cleavage study on plasmid DNA