Background: Drug resistance is the major problem for all of the currently available antifungals and, therefore, a completely novel class of antifungal compounds is an urgent need. The aim of this study is to investigate whether appropriately functionalized cationic dendritic polymers that have extensively been studied as drug delivery systems and/or antibacterial agents exhibit fungicidal activity and thus could be used as antifungals. Methods: In this work, hyperbranched polyethyleneimine and its end-group functionalized guanidinylated or quaternized derivatives were synthesized and their antifungal activity was evaluated against the opportunistic pathogen Aspergillus nidulans (A. nidulans) as a model eukaryotic microorganism. Specifically, their subcellular localization in germlings originating from fungal conidiospores, their effect on fungal growth and their ability to cause cell death in both hyphae and quiescent conidia, were studied using confocal laser and epifluorescent microscopy, the colony forming unit (CFU) assay and the propidium iodide (PI) staining assay. Results: It was found that polyethyleneimine (PEI) was the most effective compound causing complete growth inhibition at relatively lower minimum inhibitory concentration (MIC) of ca. 2 μg/mL compared to the guanidinylated (GPEI) (3 μg/mL) or quaternized (QPEI) (5 μg/mL) derivatives. More importantly, the compounds prevent quiescent conidiospore germination and, therefore, the formation of hyphae and mycelium. Employing the fluorescently-labeled derivatives, it was evident that the compounds are initially localized in the plasma membrane of both quiescent conidiospores and hyphae, while subsequently enter the cytoplasm, without showing localization in specific subcellular organelles. Appropriate staining with propidium iodide (PI) revealed that the membrane of cells, grown in the presence of all tested polymers at concentrations above the minimum inhibitory concentration (MIC), exhibits increased permeability to the dye, suggesting that the primary cause of cell death is the disruption of the plasma membranes integrity. Furthermore, by employing the CFU assay it was evident that a significant percentage of fungal conidiospores exhibited inability to recover growth following treatment with the examined compounds and that their survival was reduced by up to 70% (p < 0.001). Conclusions: Our results showed that the cationic hyperbranched polymeric compounds PEI and its derivatives GPEI and QPEI exhibited apparent fungicidal activity on A. nidulans cells. In addition, all compounds showed negligible in vitro cytotoxicity against three mammalian cell lines in this concentration range. Overall, our results suggest that the investigated polymeric compounds are promising antifungals and their activity deserves further investigation especially against drug-resistant fungal cells.