STAT3 is a cytoplasmic transcription factor that, once activated, conveys extracellular signals to the nucleus. Activation is achieved through Tyr705 phosphorylation that induces dimerization, nuclear translocation and transcriptional activation of target genes. STAT3 can also be phosphorylated on Ser727. Moreover, its hyper-activation has been implicated in several human cancers. Therefore, STAT3 is a good target candidate for development of anticancer drugs. STAT3 inhibitors are usually divided into indirect inhibitors, acting on upstream pathways, and direct inhibitors, directly binding to STAT3. The aim of this work was to investigate the mechanisms of action of different direct STAT3 inhibitors and define a common mode of action. All compounds showed a reduction of pTyr705 and pSer727 as well as an inhibition of proliferation and clonogenicity of cancer cells, although at different concentrations. These results were consistent with a direct binding of the drugs to STAT3-SH2 domain, confirmed by computational models and site-directed mutagenesis.