To isolate and characterize an antifungal protein from the culture broth of the bacterium Bacillus amyloliquefaciens. The antifungal protein designated as baciamin was isolated and exhibited a molecular mass around 50 kDa. Baciamin manifested a broad spectrum of antifungal activity. Baciamin could induce membrane permeabilization of tested fungi. Its antifungal activity was retained after incubation with trypsin and EDTA. Various ions tested did not affect its antifungal activity. Baciamin reduced the activity of HIV-1 reverse transcriptase (RT). It also inhibited proliferation of hepatoma, breast cancer and colon cancer cell lines. Baciamin augmented nitric oxide production by mouse macrophages. Bacillus amyloliquefaciens produces a broad-spectrum antifungal protein, baciamin. It induces membrane permeabilization in fungi but not in rabbit erythrocytes. Its antifungal activity is relatively thermostable, pH- and trypsin-stable. It demonstrates antiproliferative activity towards various tumour cells, nitric oxide-inducing activity towards macrophages, and inhibitory activity towards HIV-1 RT. Baciamin represents one of the few bacterial antifungal proteins reported to date. Most of the previously isolated antifungal molecules of bacterial origin are either peptides or ring compounds. Baciamin also exhibits other exploitable activities such as antitumour and immuno-enhancing activities.