Rhabdomyosarcoma (RMS) is the most commonly occurring malignant soft tissue tumor in children. Despite improving its treatment methods, the current outcome in the advanced stages of this tumor is not satisfactory. RMS cells are characterized by abnormal cellular signaling due to the changes in the activity of the tyrosine kinases. Thus, substances blocking the mitogenic signal transmitted by receptors with tyrosine kinase activity raise hopes for inhibition of the uncontrolled cell growth. In this study, we examined the anticancer activity of tyrphostin AG1296, a tyrosine kinase inhibitor that binds to the intracellular domain of the PDGF (platelet-derived growth factor) receptor in human RMS alveolar and embryonal cell lines. We have discovered that tyrphostin AG1296 completely inhibited cell proliferation and effectively inhibited cell viability. Tyrphostin AG1296 induced apoptosis of the RMS cells and significantly inhibited their migration. Additionally, investigated inhibitor slightly inhibited expression of AKT and phosphorylation of ERK in alveolar RMS cells. Importantly, the inhibitor exerted also potent effects on the nanomechanical properties and cytoskeleton organization of RMS cells. To conclude, tyrphostin AG1296 is a promising compound in the treatment of alveolar RMS. Undoubtedly, a better knowledge of receptor pathomechanism of tyrosine kinases may contribute to developing new, more effective ways of RMS treatment.