Ethnopharmacological relevance The water decoction of Combretum aculeatum aerial parts is traditionally used in Senegal to treat tuberculosis (TB). The extract shows significant antimycobacterial activity in a validated single-cell infection assay. Aim of the study The main aim of this study was to identify the antimycobacterial compounds in the water decoction of Combretum aculeatum. Since the traditional preparations are used orally, a bioactivity assessment of the possible bioavailable human metabolites was also performed. Materials and methods The Combretum aculeatum water decoction extract was first fractionated by flash chromatography. The fractions were submitted to an antibiotic assay against Mycobacterium marinum and to a single-cell infection assay involving Acanthamoeba castellanii as a host. Using these approaches, it was possible to correlate the antimycobacterial activity with two zones of the chromatogram. In parallel with this liquid chromatography (LC)-based activity profiling, high-resolution mass spectrometry (UHPLC-HRMS/MS) revealed the presence of ellagitannin (Et) derivatives in the active zones of the chromatogram. Isolation of the active compounds was performed by preparative chromatography. The structures of the isolated compounds were elucidated by nuclear magnetic resonance (NMR). Additionally, the main human metabolites of commercially available Ets were biologically evaluated in a similar manner. Results The in vitro bioassay-guided isolation of the Combretum aculeatum water extract led to the identification of three Ets (1–3) and ellagic acid (4). The major compounds 2 and 3 (α- and β-punicalagin, respectively), exhibited anti-infective activity with an IC50 of 51.48 μM. In view of the documented intestinal metabolism of these compounds, some metabolites, namely, urolithin A (5), urolithin B (6) and urolithin D (7), were investigated for their antimycobacterial activity in the two assays. Urolithin D (7) exhibited the strongest anti-infective activity, with an IC50 of 345.50 μM, but this was moderate compared to the positive control rifampin (IC50 of 6.99 μM). The compounds assayed had no observable cytotoxicity towards the amoeba host cells at concentrations lower than 200 μg/mL. Conclusion The observed antimycobacterial properties of the traditional water decoction of Combretum aculeatum might be related to the activity of Ets derivatives (1–3) and their metabolites, such as ellagic acid (4) and urolithin D (7). Despite the relatively weak activity of these metabolites, the high consumption of tannins achieved by taking the usual traditional decoction doses should lead to an important increase in the plasmatic concentrations of these active and bioavailable metabolites. These results support to some extent the traditional use of Combretum aculeatum to treat tuberculosis.