Abstract Duroia saccifera (Rubiaceae) occurs in the Amazon rainforest and their extracts showed antibacterial properties. To obtain greater quantities of active substances, leaf segments from in vitro D. saccifera seedlings were used as explants for calli induction; calli were multiplied via multiple subcultures, dried and extracted with hexane followed by ethyl acetate (EtOAc) and methanol (MeOH). As D. macrophylla had been reported to produce antimycobacterial substances, we assayed calli extracts against Mycobacterium tuberculosis (H37Rv strain). Calli EtOAc extract was active, with a minimal inhibitory concentration (MIC) of ≤ 25 mg mL-1, IC90of 19.5 mg mL-1 and minimal bactericidal concentration (MBC) of 200 mg mL-1. EtOAc extract was analyzed by Thin Layer Chromatography (TLC) and Nuclear Magnetic Resonance (NMR) to determine its chemical profile, and was found to be rich in terpenes. Chromatographic fractionation of the EtOAc extract yielded a mixture of two sterols, β-sitosterol and stigmasterol (in proportion of 2:1), which were identified by 1H and 13C NMR analysis. As far as we know, this is the first report of Duroia saccifera in vitro cell culture, antituberculosis activity of calli extract and β-sitosterol and stigmasterol isolation from in vitro plant cell culture.