Luteolin is a common flavonoid that exists in many types of plants and exerts antioxidant and anti-inflammatory activity. Luteolin presents several hydroxyl groups, which limits their applications in some fields, due to their low solubility in lipophilic systems. To avoid that, luteolin can be acylated by lipase, using vinyl ester as acyl-donor. The aim of this study was the cytotoxicity evaluation of the non-modified luteolin and its comparison with the corresponding action of the reaction mixture after enzymatic modification with Thermomyces lanuginosus lipase (Luteolin-TLL) (3΄-O-acetyl luteolin and 4΄-O-acetyl luteolin), as well as the isolated fraction (3΄-O-acetyl luteolin). The NIH/3T3 fibroblasts were used for the in vitro experiments. Cytotoxicity was estimated by means of the MTT and clonogenic assay and flow cytometry was applied for the detection of Reactive Oxygen Forms (ROS) and cell cycle analysis. The compounds showed a dose- and time-dependent cytotoxic effect against NIH/3T3 cells. Long-term toxicities of luteolin and luteolin-TLL were higher than their short-term toxicities, while the isolated fraction with high short-term toxicity had lower long-term toxicity. Treatment with 10 μg/mL luteolin-TLL or the isolated product resulted in a mild increase in S-phase. On the contrary, luteolin arrested proliferating NIH/3T3 cells at Go/G1 phase. Luteolin-TLL and the isolated compound presented a greater ability to scavenge intracellular ROS. Enzymatic modification with TLL differentiated luteolin’s biological effects especially long-term cytotoxicity against the normal cells. Nonetheless, further molecular experiments will unfold more details about the compound’s mechanism of action.