Objective: The rise of antibiotic resistance among bacterial pathogens poses a critical threat to modern medicine, driving the need for novel chemical scaffolds to develop antibacterial drugs with original mechanisms of action. Although arylidene-imidazolones, which form the structural core of the green fluorescent protein (GFP) chromophore, are widely used in bioimaging, their potential as drug precursors remains poorly understood. This work aimed to screen a library of 584 arylidene-imidazolone derivatives to identify compounds targeting bacterial replication and translation. Methods: Primary screening was conducted using the pDualRep2 reporter system and antibiotic-hypersensitive Escherichia coli strains. This approach enabled the simultaneous assessment of antibacterial activity and the collection of preliminary data on the mechanism of action. Based on these results, six candidate compounds were selected for subsequent mechanistic evaluation. Results and discussion: It was established that compounds I and II inhibit DNA polymerase I, whereas compounds IV–VI suppress bacterial translation in a cell-free system. Conclusions: The obtained results demonstrate that the arylidene-imidazolone framework represents a promising scaffold for the development of novel antibacterial agents.