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This report aims to characterize and reuse fish head powder (FHP) as a potential carbon/nitrogen source for microbial fermentation to produce prodigiosin (PG), and to evaluate its novel anti-fungal effect via an experimental and docking study. FHP has high protein (30.25%) and lipid (5.12%) content and a diversity of elements, which suggests that FHP is a potential substrate for microbial fermentation. <i>Serratia marcescens</i> TNU01 was screened as the most effective strain to produce PG from FHP. Further experiments indicated that the combination of FHP and crude chitin at the ratio of 9/1 and used at 1.5% as a C/N source for fermentation significantly enhanced PG production. Supplementing a minor amount of 0.05% Ca₃(PO₄)₂, 0.03% K₂SO₄ also positively affects PG yield production. The fermentation using a bioreactor system (type of 14 L) achieved the highest PG yield production (6.4 mg/mL) in a short cultivation time (10 h) in comparison with the highest PG yield (5.8 mg/mL) obtained after 36 h of fermentation in a flask. In the anti-fungal evaluation of various plant pathogen fungal strains, PG demonstrated efficacy against <i>Fusarium solani</i> F04. The molecular docking results also showed that PG effectively interacted with targeting protein 3QPC of <i>F. solani</i> with good binding energy (DS—9.2 kcal/mol) and an acceptable RMSD value (0.94 Å). In this study, we provide a novel application of FHP for the eco-friendly production of PG, which can potentially used for the management of <i>Fusarium solani</i>.