Polyacrylonitrile (PAN)‐based antimicrobial polymeric microspheres were synthesized and characterized in the present study. In first approach, hydrolysis of PAN microsphere was performed in alkaline medium to generate carboxylic groups, followed by iodination to obtain iodine incorporated hydrolyzed polymeric microspheres. In second approach, pendant nitrile groups of the PAN microspheres were reduced by lithium aluminum hydride to generate amino functionality. Incorporation of antimicrobial agent was achieved by reacting glycidyltrimethylammonium chloride onto the amine‐terminated microspheres. Polymeric samples obtained by both approaches were characterized by analytical, morphological, and spectral techniques. Hydrolyzed microspheres showed higher aqueous swelling capacity, absorbing almost 2000% water in 10–12 days. Both iodinated hydrolyzed polyacrylonitrile (I2HPAN) and quaternized polyacrylonitrile (QPAN) microspheres showed prominent zone of inhibition against both gram‐positive (Staphylococcus aureus) and gram‐negative (Escherichia coli) bacteria. Concentration and time‐dependent bacterial mitigation was observed when different amounts of both I2HPAN and QPAN microspheres were mixed with bacterial solution. I2HPAN established higher killing efficacy than QPAN. PAN‐based antimicrobial microspheres have strong potential to be used as broad spectrum antimicrobial matrices insoluble for water disinfection. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47968.