The principle aim of this study was to evaluate the anti-biofilm activity of newly synthesized hexahydroimidazo [1,5-a] pyridinium bromides with varying benzyl substituents to test as biofilm eradication agents against 10 opportunistic pathogens. To our knowledge this important aspect of their anti-biofilm and antioxidant activity has not yet been discussed in the literature. Anti-biofilm activity of benzyl substituted hexahydroimidazo[1,5-a]pyridinium bromide salts (B1-B9) was tested accoriding to microtitre plate method. Additionally, the best biofilm production of E.coli ATCC 8739 was monitoring with SEM (Scanning Electron Microscopy). The antioxidant activity of these salts were studied by the method of ABTS (2,2 azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation. In this study we have shown that these compounds possess excellent, broad spectrum antibiofilm activity against opportunistic microorganisms: the bacteria Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Micrococcus luteus, Proteus vulgaris, Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae and the yeasts Candida albicans, Candida krusei. All of these compounds inhibited bacterial and fungal biofilm formation at concentrations ranging from 0.78 to 400 mu g/mL, 3.12 to 50 mu g/mL, respectively (biofilm inhibitory concentration=BIC). Additionally, these compounds have been found to possess antioxidant potential with the highest inhibitory percentage of 92.4% using ABTS radical cation assay. The obtained results revealed that all the salts displayed anti-biofilm and antioxidant activity against the tested microorganisms, indicating that these salts could use in the design of new therapeutics and more effective antimicrobials for the control and prevention of biofilm infections caused by the spread of pathogenic microorganisms to the environment.