The emergence of antibiotic-resistant pathogenic bacteria is a healthcare problem worldwide. We evaluated the antimicrobial activity of rhodomyrtone, an acylphloroglucinol present in Rhodomyrtus tomentosa leaves, against the human Gram-positive pathogen Streptococcus pneumoniae. The compound exhibited pronounced anti-pneumococcal activity against a broad collection of clinical isolates. We studied the effects at the molecular level by integrated proteomic and metabolomic analysis. The results revealed alterations in enzymes and metabolites involved in several metabolic pathways including amino acid biosynthesis, nucleic acid biosynthesis, glucid, and lipid metabolism. Notably, the levels of two enzymes (glycosyltransferase and UTP-glucose-1-phosphate uridylyltransferase) and three metabolites (UDP-glucose, UDP-glucuronic acid and UDP-N-acetyl-D-galactosamine) participating in the synthesis of the pneumococcal capsule clearly diminished in the bacterial cells exposed to rhodomyrtone. Rhodomyrtone-treated pneumococci significantly possessed less amount of capsule, as measured by a colorimetric assay and visualized by electron microscopy. These findings reveal the utility of combining proteomic and metabolomic analyses to provide insight into phenotypic features of S. pneumoniae treated with this potential novel antibiotic. This can lead to an alternative antibiotic for the treatment of S. pneumoniae infections, because of the growing concern regarding antimicrobial resistance.