Aeroponically grown <i>Physalis acutifolia</i> afforded five new and six known withanolides including 10 physalins. The structures of the new withanolides, acutifolactone (<b>1</b>), 5β,6β-epoxyphysalin C (<b>2</b>), 5α-chloro-6β-hydroxyphysalin C (<b>3</b>), and an inseparable mixture of 5β,6β-epoxy-2,3-dihydrophysalin F-3β-<i>O</i>-sulfate (<b>4</b>) and 5β,6β-epoxy-2,3-dihydrophysalin C-3β-<i>O</i>-sulfate (<b>5</b>), were elucidated by analysis of their spectroscopic data and chemical interconversions. The known withanolides were identified as physalins B (<b>6</b>), D (<b>7</b>), F (<b>8</b>), H (<b>9</b>), I (<b>10</b>), and U (<b>11</b>) by comparison of their spectroscopic data with those reported. Evaluation of <b>1</b>-<b>11</b> and the derivatives, <b>13</b> and <b>13a</b>, obtained from <b>4</b> and <b>5</b> against a panel of four human cancer cell lines [NCI-H460 (non-small-cell lung), SF-268 (CNS glioma), PC-3 (prostate adenocarcinoma), and MCF-7 (breast adenocarcinoma)] and normal human lung fibroblast (WI-38) cells revealed that physalins <b>2</b>, <b>3</b>, <b>8</b>, and <b>9</b> exhibited selective cytotoxic activity to at least one of the cancer cell lines tested compared to the normal cells and that <b>7</b>, <b>10</b>, and <b>11</b> were inactive up to a concentration of 10.0 μM. These data provided some preliminary structure-activity relationships and suggested that the mechanism of cytotoxic activity of physalins may differ from other classes of withanolides.