Drug-resistant pathogens pose severe threats to public health, driving the need for new antibacterial agents. Herein, we report the design, synthesis, and biological evaluation of a series of 1,3-diamino-7H-pyrrolo[3,2-f]quinazoline derivatives (PQDs) as dual-targeting antimicrobials acting on dihydrofolate reductase (DHFR) and membrane integrity. In vitro assays showed most compounds exhibited broad-spectrum activity, with MIC values as low as 0.0625 μg/mL. Compound 4d (7-(3,4,5-trifluorobenzyl)-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine) stood out, exhibiting 8-fold greater activity than the lead compound IRS-16 against all tested strains, lower toxicity to LO2 cells than 5-fluorouracil, and superior bactericidal efficacy to norfloxacin. It also displayed reduced resistance propensity (minimal MIC increase over 20 days vs. 8- to 32-fold for norfloxacin). Furthermore, compound 4d strongly inhibited ecDHFR (IC50 = 0.75 nM vs. TMP's 147.1 nM), disrupted bacterial inner membranes, inhibited biofilm formation, and attenuated phage-related processes. Multi-omics analysis further confirmed that compound 4d exerts its antibacterial effects through multiple mechanisms, including synergistic DHFR inhibition, disruption of cell membrane integrity, and interference with amino acid and nucleotide metabolic pathways. In acute toxicity tests in mice, 4d showed a higher LD50 (17.39 mg/kg) than IRS-16 (14.32 mg/kg), indicating better safety. These findings demonstrate that compound 4d is a promising dual-targeting antibacterial candidate for further development.