A unique class of antibacterial azolylpyrimidinediols (APDs) and their analogues were developed. Some synthesized compounds showed excellent bacteriostatic potency; especially, triazolylpyrimidinediol (triazolyl PD) 7a exhibited good anti-Acinetobacter baumannii potential with a low MIC of 0.002 mmol/L. Triazolyl PD 7a with inconspicuous cytotoxicity and hemolytic activity could eradicate the established biofilm, showed low resistance, and exhibited favorable drug-likeness. Mechanistic explorations revealed that compound 7a without membrane-targeting ability could decrease metabolic activity, interact with DNA through groove binding action to block DNA replication rather than intercalate into and cleave DNA, and thus inhibit bacterial growth. Further computations displayed that the low EHOMO and large energy gap might help triazolyl PD 7a binding to biological targets more easily. Moreover, compound 7a gave appreciable in vivo pharmacokinetic properties and pharmacodynamics. These findings of azolylpyrimidinediols as novel structural scaffolds of DNA-groove binders might imply a large promise for the treatments of Acinetobacter baumannii infection.