Hyperbranched polyethyleneimine having 25,000Da molecular weight was functionalized by a simple sulfopropylation reaction, affording a novel N‐sulfopropylated PEI derivative (PEI‐SO3‐). The successful introduction of N‐sulfopropyl and sulfobetaine groups to the amino groups of PEI was spectroscopically confirmed. Furthermore, the antibacterial and anti‐cyanobacterial activity of PEI‐SO3‐ in comparison to the parent PEI were investigated on two type heterotrophic bacteria, i.e., Gram (‐) Escherichia coli and Gram (+) Staphylococcus Aureus bacteria, and one type of autotrophic cyanobacterium, i.e. Synechococcus sp. PCC 7942. Both PEI‐SO3‐ and PEI showed an enhanced, concentration‐dependent antibacterial and anti‐cyanobacterial activity against the tested bacteria strains, with PEI‐SO3‐ exhibiting higher activity than the parent PEI, signifying that the introduction of the sulfopropyl and sulfobetaine groups to the PEI amino groups enhanced the antibacterial anti‐cyanobacterial properties of PEI. In the case of cyanobacteria, PEI‐SO3‐ was found to affect the integrity of the photosynthetic system by the inhibition of Photosystem‐II electron transport activity. Cytocompatibility and hemocompatibility studies revealed that PEI‐SO3‐ exhibits high biocompatibility, suggesting that PEI‐SO3‐ could be considered as an attractive antibacterial and anti‐cyanobacterial candidate for various applications in the disinfection industry and also against the harmful cyanobacterial blooms.