The antibacterial, bactericidal and anti-biofilm activity of pyridyl tetrazole derivatives were evaluated. In vitro antibacterial activity was investigated by microdilution method against both Gram-positive and Gram-negative bacterial strains. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) have been determined and found to be in the range of 3.9 to 15.6 μg ml−1. It was that two test compounds 3D and 4D were more promising against Gram-negative bacteria with an MIC value as low as 3.9 μg ml−1 against E. coli MTCC 739. Anti-biofilm activity was investigated by crystal violet assay where M. luteus MTCC 2470, Escherichia coli MTCC 739 and Klebsiella planticola MTCC 530 were used as the test organisms. The half maximal biofilm inhibitor concentration (BIC50) was determined and found to be in the range of 10.3 to 17.3 μg ml−1.