According to the World Health Organization (WHO), cancer is the second cause of death globally. Conventional cancer therapy includes surgery, chemotherapy, and radiotherapy. Nevertheless, therapy is often limited by low efficacy or significant adverse side effects. Therefore, safer and more efficient therapeutic agents are essential. Flavonoids, compounds largely found in the plant kingdom, have shown promising cancer-inhibiting properties. Although the cytotoxic effect of flavonoids in human cancer cell lines is widely reported, the corresponding effect in healthy human cells is underreported. The present study aims to evaluate the toxicity of a group of flavonoids hydroxylated at C-7, C-3′, and C-4′ in lung fibroblasts. To achieve this, the MRC-5 human lung fibroblast cell line was incubated with flavonoids, 0–160 µM, with additional hydroxy groups at C-3, C-6, or C-5′, or chlorine at C-3. After incubation for 48 h, the inhibition of cell viability and growth was measured using WST-8 and sulforhodamine B assays, respectively. The presence of the 3- or 5-hydroxy groups was associated with lower cytotoxicity at low concentrations (40 µM); meanwhile, at higher concentrations (>40 µM), only the presence of the 5-hydroxy group seemed to be related to low cytotoxicity. Although additional studies are required, these results reveal substituted flavonoids with lower in vitro toxicity in healthy human cells.