Four couples of 5,6-membered bis(metallacyclic) Pt(II) complexes with acetylide and isocyanide auxiliary ligands have been prepared and characterized. The structures of (-)-2 and (-)-3 are confirmed by single-crystal X-ray diffraction, showing a distorted square-planar coordination environment around the Pt(II) nucleus. Both solutions and solid samples of all complexes are emissive at RT. Acetylide-coordinated Pt(II) complexes have a lower energy emission than those isocyanide-coordinated ones. The emission spectra of N^N'*C-coordinated Pt(II) derivatives show a lower energy emission maximum relative to N^C*N'-coordinated complexes with the same auxiliary ligand. Moreover, the difference between cyclometalated N^N'*C and N^C*N' ligands exerts a more remarkable effect on the emission than the auxiliary ligands acetylide and isocyanide. Cytotoxicity and cell imaging of luminescent 5,6-membered bis(metallacyclic) Pt(II) complexes have been evaluated.