World health organization listed fungi as priority pathogens in 2022 to counter their adverse effects on human well-being. The use of antimicrobial biopolymers is a sustainable alternative to toxic antifungal agents. In this study, we explore chitosan as an antifungal agent by grafting a novel compound N-(4-((4-((isatinyl)methyl)piperazin-1-yl)sulfonyl)phenyl) acetamide (IS). The acetimidamide linkage of IS to chitosan herein was confirmed by ¹³C NMR and is a new branch in chitosan pendant group chemistry. The modified chitosan films (ISCH) were studied using thermal, tensile, and spectroscopic methods. The ISCH derivatives strongly inhibit fungal pathogens of agricultural and human importance, namely Fusarium solani, Colletotrichum gloeosporioides, Myrothecium verrucaria, Penicillium oxalicum, and Candida albicans. ISCH80 showed an IC₅₀ value of 0.85 μg/ml against M. verrucaria and ISCH100 with IC₅₀ of 1.55 μg/ml is comparable to the commercial antifungal IC₅₀ values of Triadiamenol (3.6 μg/ml) and Trifloxystrobin (3 μg/ml). Interestingly, the ISCH series remained non-toxic up to 2000 μg/ml against L929 mouse fibroblast cells. The ISCH series showed long-standing antifungal action, superior to our lowest observed antifungal IC₅₀ values of plain chitosan and IS at 12.09 μg/ml and 3.14 μg/ml, respectively. ISCH films are thus suitable for fungal inhibition in an agricultural setting or food preservation.