Abstract Nanoparticles and nanomaterials are revolutionizing medicine by offering diverse tools for diagnosis and therapy, including devices, contrast agents, drug delivery systems, adjuvants, therapeutics, and theragnostic agents. Realizing full applied potential requires a deep understanding of the interactions of nano dimensional objects with biological cells. In this study, we investigate interaction of single-crystal diamond nanoneedles (SCDNNs) containing silicon vacancy (SiV-) color centers with biological substances. Four batches of the diamond needles with sizes ranging between 200 nm and 1300 nm and their water suspensions were used in these studies. The human lung fibroblast cells were used for the proof-of-concept demonstration. Employing micro-photoluminescence (PL) mapping, confocal microscopy, and lactate dehydrogenase (LDH) viability tests, we evaluated the cellular response to the SCDNNs. Intriguingly, our investigation with PL spectroscopy revealed that the cells and SCDNNs can coexist together with approved efficient registration of SiV- centers presence. Notably, LDH release remained minimal in cells exposed to optimally sized SCDNNs, suggesting a small number of lysed cells, and indicating non-cytotoxicity in concentrations of 2–32 µg ml−1. The evidence obtained highlights the potential of SCDNNs for extra- or/and intracellular drug delivery when the surface of the needle is modified. In addition, fluorescent defects in the SCDNNs can be used for bioimaging as well as optical and quantum sensing.