Five new tripeptides, acidiphilamides A-E (1-5), were discovered along with two previously reported compounds, l-isoleucinamide (6) and l-valinamide (7), from Streptacidiphilus rugosus AM-16, an acidophilic actinobacterial strain isolated from acidic forest soil. The structures of 1-5 were elucidated as modified tripeptides bearing phenylalaninol or methioninol fragments with C3-C5 acyl chains based mainly on NMR and mass spectroscopic data. The absolute configurations of the amine units were established by advanced Marfey's method and GITC (2,3,4,6-tetra- O-acetyl-β-d-glucopyranosyl isothiocyanate) derivatization followed by LC/MS analysis. Acidiphilamides A and B (1 and 2), the first secondary metabolites isolated from the rare actinobacterial genus Streptacidiphilus, significantly inhibited autophagic flux but not proteasome activity in HeLa cells. These compounds appeared to block mainly the autophagosome-lysosome fusion step in the late stage of cellular autophagy.