Phytochemical investigation of the stem bark of Stereospermum acuminatissimum resulted in the isolation of seventeen compounds, including four tirucallanes (1–4), three hopanes (5–7), one oleanane (8), three sterols (9–11), three saponins (12–14), two cinnamic acids (15–16), and one iridoid (17). The dehydration of compound 5 led to compound 5a. The structures of all compounds were elucidated by analysis of spectroscopic and mass spectrometric data. Compounds 1, 6 and 7 are reported from the Bignoniaceae family for the first time, while compounds 11–14 and 16 are reported from S. acuminatissimum for the first time. The in vitro antibacterial activities of some compounds (1–5, 8, 5a), the crude extract and the ethyl acetate fraction were assessed. The crude extract showed significant antibacterial activity against Staphylococcus aureus, Salmonea pneumonia and Klebsiella pneumoniae with MIC values of 62 μg/mL. Compound 1 revealed moderate antibacterial activities against K. pneumoniae, Shigella flexneri and Methycillin resistant S. aureus with MIC values of 62, 62 and 31 μg/mL respectively.