Microbial infections are leading causes of death and morbidity all over the world due to the development of the resistance to antibiotics by certain microorganisms. In this study, the chemical exploration of the ethanol (EtOH) extract of the aerial part of Dracaena stedneuri (Dracaenaceae) led to the isolation of one previously unreported chalcone derivative, i.e., 2′,4′-dihydroxy-2,3′-dimethoxychalcone (1), together with 12 known compounds: 8-(C)-methylquercetagetin-3,6,3′-trimethyl ether (2), methylgalangine (3), quercetin (4), kaempferol (5), 6,8-dimethylchrysin (6), ombuine-3-O-rutinoside (4ʹ,7-dimethylquercetin-3-O-α-L-rhamnopyranosyl-(1 → 6) -β-D-glucopyranoside) (7), alliospiroside A (8), β-sitosterol 3-O-glucopyranoside (9), ishigoside (10), betulinic acid (11), oleanolic acid (12), and lupeol (13). The structures were determined by spectroscopic and spectrometric analysis including 1- and 2-Dimensional Nuclear Magnetic Resonance (1D- and 2D-NMR), High-Resolution Electrospray Ionization Mass Spectrometry (HRESIMS), and comparison with literature data. The isolated secondary metabolites and crude extract displayed antibacterial activity against some multidrug-resistant strains with minimal inhibitory concentration (MIC) values ranging from 32 to 256 μg/mL. The antibacterial activity of compound 13 against Enterobacter aerogenes ATCC13048 (MIC value: 32 μg/mL) was higher than that of chloramphenicol used as the reference drug (MIC = 64 μg/mL).