Fungal bioluminescence is a genetically encodable platform with potential for bioimaging in mammalian systems but remains limited by low signal intensity. Here, we report a series of fungal luciferin analogs designed by modification of the arylidene fragment. Electron-donating substituents generally increase activity, whereas extension of π-conjugation red-shifts emission, with partial decoupling of these effects. These trends parallel those observed for firefly luciferins, indicating that empirical design principles can be transferred between the two systems. Evaluation across fungal luciferases and engineered variant v4, demonstrated broad substrate compatibility and highlighted the combined potential of luciferase engineering and luciferin modification for improving fungal bioluminescence in mammalian bioimaging.