PARP1 is a nuclear enzyme involved in DNA repair processes. Since its inhibition causes sensitization to DNA damaging chemotherapy (by the so-called “synthetic lethality”), several inhibitors have been recently developed and exploited for clinical use. However, the emergence of resistance to PARP1 inhibitors increased the interest towards alternative approaches able to interfere with PARP1 activity. In particular, within the promoter region of PARP1 a characteristic, non-canonical (3+1) G-quadruplex-forming sequence was identified. A strong correlation between G-quadruplex stabilization in gene promoters and transcriptional regulations has been proposed for several oncogenes. Since no PARP promoter modulators have been described so far, the interaction with a small collection of G-quadruplex binders was investigated, taking into account the particular hybrid topology of PARP1 G-quadruplex. Six structurally diverse compounds, extensively studied and known for showing great affinity towards canonical G-quadruplex, were selected, and NMR, CD, and fluorescence titration studies were carried out. The results from the physico-chemical analyses, confirmed by molecular modelling, showed that only one of the tested compounds showed strong stabilization of the nucleotide, demonstrating that the structural requirements for an optimal interaction between each of the ligands and the peculiar hybrid G-quadruplex region are quite strict. Overall, the studied compounds can be considered as a starting point for the identification of the key features necessary for a selective interaction with the PARP1 promoter G-quadruplex.