The fungus growing termite species Macrotermes bellicosus (M. bellicosus) is used in nutrition and traditional medicine in the Republic of Benin for the treatment of infectious and inflammatory diseases. Previous findings demonstrated evidence of anti-inflammatory and spasmolytic properties of M. bellicosus. The aim of the present study was to evaluate the antimicrobial potential of different extracts of M. bellicosus samples and determine the chemical profile of an ethanolic M. bellicosus extract. Chemical profiling was conducted using centrifugal partition chromatography and 13C-NMR, followed by MALDI-TOF MS. Major identified compounds include hydroquinone (HQ), methylhydroquinone (MHQ), 3,4-dihydroxyphenethyl glycol (DHPG), N-acetyldopamine (NADA) and niacinamide. The fatty acid mixture of the extract was mainly composed of linoleic and oleic acid and highlights the nutritional purpose of M. bellicosus. Using the Kirby–Bauer disc diffusion and broth microdilution assay, an antibacterial activity of M. bellicosus samples was observed against various clinical strains with a highest growth inhibition of S. aureus. In addition, HQ and MHQ as well as fractions containing DHPG, niacinamide and NADA inhibited S. aureus growth. The reported antimicrobial activity of M. bellicosus and identified active substances provide a rationale for the traditional medicinal use of M. bellicosus.