A new acylphloroglucinol with a novel architecture including an unprecedented dearomatic dibenzofuran core, named callistemenonone A (1), was isolated from the leaves of Callistemon viminalis (Myrtaceae). The structure was fully characterized on the basis of extensive spectroscopic analysis, including UV, HRESIMS, as well as 1D and 2D NMR spectral data (HSQC, HMBC, and ROESY). The deduced structure represents the first example of a natural dibenzofuran with two phenyl moieties coupling through tertiary hydroxy and ketal carbons. A plausible biogenetic pathway involving oxidative coupling and dearomatization as key steps is proposed to account for the biosynthesis of this novel class of dibenzofuran. Moreover, antimicrobial assays, in conjunction with the time-killing and biophysical studies, revealed that 1 exerted potent bactericidal activity against a panel of methicillin resistant pathogenic microbes with a unique mechanism.