Genome functions are regulated by local chromatin structure and also by the association of individual genes with nuclear structures. As candidates for involvement in functional chromatin and nuclear organization, we investigated the actin family, which consists of conventional actin and actin-related proteins (Arps). Interestingly, a portion of actin and some Arp subfamilies are localized in the cell nucleus. Monomeric G-actin and the nuclear Arps are known to contribute to genome functions (including transcription and DNA damage repair) as components of chromatin remodeling complexes and histone modification complexes. In addition to nuclear G-actin, nuclear actin filament (F-actin) is also involved in genome functions and nuclear organization. Although a sufficient amount of actin is found in the nucleus, information regarding factors involved in regulating the formation of nuclear F-actin is still limited. We showed that Arp4 (one of the nuclear Arps) is a suppressor of nuclear F-actin formation, which suggests that crosstalk between actin family proteins in the nucleus performs important roles in chromatin functions and nuclear organization. For further analysis and operation of nuclear actin family proteins, we screened and evaluated bicyclic peptides binding to these molecules. Bicyclic peptides contain two macrocyclic rings, and this structure contributes to high affinity binding to target molecules. We performed screening of bicyclic peptide libraries by the phage display technique, and obtained bicyclic peptides for nuclear Arps and G-actin. We introduced these bicyclic peptides into living cells by electroporation, and evaluated the peptides. Bicyclic peptides for Arp5 and Arp8 (components of the INO80 chromatin remodeling complex) inhibited functions of INO80 complex in the living cells. We also successfully delivered bicyclic peptides for G-actin into the nucleus by tagging a nuclear localization signal (NLS). We observed that the NLS-bicyclic peptides for G-actin suppressed nuclear F-actin formation and impaired the function of nuclear F-actin in DNA damage repair. These bicyclic peptides provide novel information on the roles of actin family proteins in the nucleus.