The anticancer activity of N-benzylisoindole-1,3-dione derivatives was evaluated against adenocarcinoma (A549-Luc). First, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide activity assay studies of two isoindole-1,3-dione derivatives were performed against A549 cell lines. Both compounds showed inhibitory effects on the viability of A549 cells. Then, we explored the potential of these compounds as active ingredients by in vivo studies. Nude mice were given A549-luc lung cancer cells, and tumor growth was induced with a xenograft model. Then, nude mice were divided into three groups: the control group, compound 3 group, and compound 4 group. After application of each compound to the mice, tumor sizes, their survival, and weight were determined for 60 days. Furthermore, toxicological studies were performed to examine the effects of the drugs in mice. In addition to toxicological studies, histopathological analyses of organs taken from mice were performed, and the results were evaluated. The obtained results showed that both N-benzylisoindole derivatives are potential anticancer agents.