In this paper, we describe the chemical structure and biological activity of nigerazine A (I), a co-metabolite of nigerazine B (II) from Aspergillus niger 1-639. Nigerazine A, obtained as a colorless oil positive to Dragendorff's reagent, showed a molecular ion peak at m/z 258 (C₁₆H₂₂N₂O) in mass spectrum, with UV and IR spectra similar to nigerazine B, suggesting it was an isomer of II. Through ¹³C-NMR (25MHz) and ¹H-NMR (100MHz) analyses at different temperatures (23°C and 80°C) and decoupling experiments, the structure of I was determined as N-methyl-cis-2,5-dimethyl-N'-cinnamoyl-piperazine (in contrast to the trans configuration of II), with the configuration of the two asymmetric carbons suggested to be (R,R) or (S,S). The temperature-dependent NMR spectra of I were interpreted by conformational mobility: at 80°C, signals from two conformers were averaged due to rapid equilibrium on the ¹³C-NMR time scale. Biological activity assays showed that I had no antimicrobial activity against Escherichia coli K-12, Bacillus subtilis, Pseudomonas putida, Staphylococcus aureus, Mycobacterium sp. 607, Candida albicans, Saccharomyces cerevisiae, and Penicillium chrysogenum at 100 mcg/ml. It reduced the root elongation of lettuce seedlings (Lactuca sativa cv. Great Lakes 366) by about 50% at 100 ppm, whereas II reduced it by about 40% at the same concentration.