Hyperbranched polyethyleneimine having 25,000 Da 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 and the 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.