Half-sandwich iridium(III) metallocomplexes represent a prospective structural type for the development of anticancer compounds. Our metallocomplexes showed high cytotoxicity in different cancer cell lines (2D and 3D cultures), including cisplatin-resistant lung carcinoma cells, while they were inactive against non-cancerous cells. For the first time, the effect of anticancer half-sandwich Ir complexes on the expression of stress-related genes associated with DNA damage and endoplasmic reticulum stress is discussed. Proteomic analysis reveals that 3 induces nucleolar stress and suppresses oxidative phosphorylation in A549 cells. This activates p53 and locks the RB/E2F axis, which is critical for initiating DNA replication. The cells are trapped before and during the S phase. The resulting cell phenotype is therefore predominantly cytostatic with apoptotic priming rather than purely genotoxic, as observed with cisplatin. The anti-cancer potential of selected metallocomplexe was proved in vivo where it affected tumor growth.