Sugarcane is Australia's second largest export crop and generates an annual revenue over $4 billion. A perennial crop grown along the eastern coast of Queensland and in northeast New South Wales, sugarcane cultivation is impacted by biotic and abiotic stresses. The use of nanomaterials to mitigate these stresses has attracted attention in recent years as they have the potential to provide more sustainable solutions. Carbon nanodots (C-dots) are carbogenic nanoparticles that are typically smaller than 10 nm, water dispersible, UV absorbing, highly fluorescent and biocompatible. C-dots have been identified as a potential nanocarrier and nutraceutical material for sugarcane farming. Therefore, evaluation of their toxicity towards sugarcane setts, human cell lines and other agriculture-relevant animals such as bees is imperative for assessing their suitability for agricultural applications. In this study, carbon dots (CD) were synthesized in-house, and different concentrations were applied to sugarcane setts at 10 min dipping times to evaluate their impact on germination, plant height, and biomass four weeks after planting (WAP). Cytotoxicity evaluation using normal human dermal fibroblast (NHDF) cells and MDA-MB-231 breast cancer cells exposed to CD showed no change in cell viability. Overall, the CDs did not exhibit phytotoxicity and cytotoxicity indicating their feasibility for use in sugarcane cultivation.