Trichorzin PA is a family of 18-residue peptaibols with linear and flexible peptide chains. The three-dimensional structures and biological activities of trichorzin PA peptaibols are largely uncharacterised. In this work, two previously identified peptaibols, trichorzin PA VI (1) and II (2), were isolated from Trichoderma lentiforme ML-P8-2. While for the first time, we report here the X-ray crystallographic structure of 1, antimicrobial activities against a panel of common pathogenic bacteria and fungi, and cytotoxicities of 1 and 2. In bioassays, 1 and 2 exhibited strong antimicrobial activities against the seven tested microbes, with MIC values in the range of 0.19–6.25 μM. Additionally, 1 and 2 displayed potent cytotoxicities against five human cancer cell lines, with IC50 values in the range of 0.01 ± 0.02–2.75 ± 0.17 μM. The bioassay results were generally better than those reported for other 18-residue peptaibols, including other trichorzin PA members.