Growth and developmental changes in plants induced by pharmaceuticals reflect changes in processes at the cellular and subcellular levels. Due to their growth and cellular characteristics, plant cell suspension cultures can be a suitable model for assessing toxicity. In this study, 10-1000 μg/L of the non-steroidal anti-inflammatory drug diclofenac (DCF) decreased the viability of Nicotiana tabacum BY-2 cells after 24 h of treatment. Further, 0.1-10 mg/L DCF diminished the density of the cell suspension by 9-46% after 96 h of treatment, but at 1 and 10 μg/L, DCF increased the density by 13% and 5%, respectively, after 120 h. These changes were accompanied by increased production of total reactive oxygen species (ROS) and mitochondrial superoxide (up to 17-fold and 5-fold, respectively), and a decrease in the mitochondrial membrane potential (by ∼64%) especially at 1000 μg/L DCF. The increased ROS production was accompanied by decrease in level of reactive nitrogen species (RNS; by 36%) and total thiols (by 61%). Damage to BY-2 cells was evidenced by accumulation of neutral red in acidic compartments (up to 10-fold at 1000 μg/L DCF), and increase of autophagic vacuole formation (up to 8-fold at 1000 μg/L DCF). Furthermore, irregular or stretched nuclei were observed in nearly 27% and 50% of cells at 100 and 1000 μg/L DCF, respectively. Highest levels of chromatin condensation (11% of cells) and apoptotic DNA fragmentation (7%) were found at 10 μg/L DCF. The results revealed a significant effect of DCF on BY-2 cells after 24 h of exposure. Changes in the growth and viability parameters were indisputably related to ROS and RNS production, changes in mitochondrial function, and possible activation of processes leading to cell death.