As of 2026, cancer remains one of the leading causes of death worldwide. Despite substantial medical advancements, chemotherapy remains widely used. However, conventional chemotherapeutic agents lack selectivity towards cancer cells and frequently induce severe side effects, including myelosuppression, gastrointestinal toxicity, and cardiotoxicity. Therefore, increasing attention has been directed towards the modulation of existing anticancer drugs through combination with metallic nanoparticles. Herein, we verified whether platinum nanoparticles (PtNPs) of defined sizes 5, 30, 50, and 70 nm interact with Mitomycin C (MMC) and one of its metabolites, 2,7-diaminomitosene (DAM). DLS and AFM analyses revealed differing aggregation patterns, with MMC exhibiting aggregation primarily under dry conditions. FTIR and NIR spectroscopies suggested that PtNPs interact with MMC predominantly through weak non-covalent interactions. Thermal inspection utilising ITC and DSC showed that these APIs interact in an endothermic manner; however, MMC data might be interpreted as negligible. Furthermore, Ames mutagenicity assay on Salmonella enterica serovar Typhimurium TA102 revealed dose-dependent reduction of mutagenic potential by PtNPs, with more pronounced effects observed for DAM. Cytotoxicity analyses in MCF-7 and SK-BR-3 cells showed that PtNP-MMC combinations generally reduced metabolic activity compared with MMC alone, although the effects varied with cell line, MMC concentration, and nanoparticle size and concentration. The response was more consistent in SK-BR-3 cells, whereas PtNPs generally attenuated DAM activity. As PtNPs alone affected metabolic activity at higher concentrations, the combination effects cannot be attributed solely to enhancement of MMC activity. These findings support further investigation of PtNPs as modulators of mitomycin-related compounds, although their mechanisms and therapeutic relevance require additional validation.