Understanding how multidrug-resistant (MDR) bacteria and fungi defy the existing antimicrobial agents requires innovative tools and techniques for real-time, in situ exploration of bacterial responses to antibiotics. Fluorescence-tagged antibiotics or dyes with inherent antimicrobial activity can provide a profound understanding of the molecular biology underlying antibiotic action and resistance mechanisms. Cutting-edge research highlights the pursuit of benzo-α-pyrone (coumarin) derivatives due to their excellent pharmacokinetics, diverse pharmacological activities, and innovative fluorescence molecular probes. In this study, donor-π-acceptor-based coumarin dyes were designed and evaluated for antimicrobial efficacy against fungal strains (Candida albicans), Gram-negative pathogens (Escherichia coli), and Gram-positive bacteria (Staphylococcus aureus). I-6 exhibited notable antimicrobial activity against S. aureus and C. albicans compared with E. coli. Conversely, I-9, a congener of I-6, showed a comparable affinity for S. aureus but found poor activity against the remaining tested strains. Mechanistic investigative studies unveiled that the inhibitory efficacy of I-6 can be attributed to its capacity to generate high reactive oxygen species (ROS) formation. Despite the evident antimicrobial potential of I-6 in the data, our future prospects, including real-time visualization to study physiological processes like uptake, distribution, and mechanism of action through fluorescence-based imaging modalities, could enhance the applicability of these probes.