Objectives: The present study aims to report on the production optimization, purification, and characterization of structural and functional attributes of a novel broad-spectrum antibacterial compound produced by Aspergillus fumigatus nHF-01 (GenBank Ac. No. MN190286).Materials and Methods: The culture conditions were optimized by using rigorous culture-set preparation considering various abiotic and biotic factors for a higher amount of antimicrobial production. The produced antimicrobial was solvent extracted and purified by preparative TLC and HPLC methods followed by characterization using UV-Vis, FT-IR, ESI-MS, and 1H-NMR spectroscopy. The MIC and MBC of the antimicrobials were determined against a set of Gram-positive and Gram-negative human pathogenic bacteria. The mode of action on cellular morphology and integrity were determined by LDH and SEM studies. Its biofilm-inhibition properties and synergistic activity with antibiotics were studied. The possible cytotoxic effect on human cell lines was also tested by MTT assay. The putative target site of action was evaluated through in silico molecular docking study. Results: The micro-fungus A. fumigatus nHF-01 produced the maximum antibacterial compound while grown in a combination of 2% MEB (w/v) and 4% YE (w/v) at pH 6.0 and 20 °C temperature with 100 rpm agitation for ten days. The DCM extractable crude compound has a potent growth inhibition against the target human food and topical pathogenic bacteria at a 15 mg/ml concentration and is stable up to 100 °C. The spectroscopic studies confirmed the antimicrobial compound as 5-butyl-2-pyridine carboxylic acid with MIC values from 0.069±0.0034 to 1.12±0.052 mg/ml and from 8.925±0.39 to 17.85±0.78 mg/ml; and MBC values from 8.925±0.40 to 17.85±0.776 mg/ml and from 0.069±0.0034 to 0.139±0.0065 mg/ml against human pathogenic Gram-positive and Gram-negative bacteria, respectively. A concentration of 0.139 and 17.85 mg/ml decreased the viability sharply within 15 min of the incubation period with the gradual increase in LDH activity, indicating a robust bactericidal and lytic mode of action. The time-kill kinetics study shows that at a 17.85 mg/ml dose (i.e. MBC), the compound caused zero viability of E. coli and S. epidermidis cells from the initial log CFU/ml 5.78 after 15 h of treatment. It caused a remarkable change in morphology like the formation of blebbing, notch, rupture of the entire cell walls, and entire dissolution of cell integrity at a concentration of 4 µg/ml and 129 µg/ml. It had cytotoxicity against the tested human lung carcinoma A549 cell line. It showed a notable antibiofilm activity at 20 µg/ml and 4 µg/ml comparable to the standard antibiofilm drug usnic acid 10 µg/ml and 64 µg/ml against E. coli and B. cereus. It had a synergistic activity with streptomycin, whereas ciprofloxacin and vancomycin showed additive effects. It showed the highest binding affinities with Quinol-Fumarate Reductase (1l0v), a respiratory enzyme. Conclusion: Thus, the above findings can be concluded that the strain A. fumigatus nHF-01 produces a novel broad-spectrum antimicrobial compound 5-butyl-2-pyridine carboxylic acid with potent bactericidal activity against human food and topical pathogenic bacteria. This is the first report of such a compound from the A. fumigatus.