Protoporphyrin IX (PpIX) is the photosensitizer in 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT). Its further bioconversion to heme requires iron and can be suppressed by iron chelators such as desferrioxamine (DFO). To investigate the effectiveness of DFO in enhancing PpIX-based PDT in skin tissue, we selected fibroblasts, HaCat cells and Hep-2 cells as targets co-cultured with ALA, that have different biological characteristics for PpIX conversion. Evaluated interventions included: (1) blank control (no ALA, no DFO); (2) DFO alone; (3) ALA alone; and (4) DFO in combination with ALA. Before photodynamic irradiation, cellular PpIX level and fluorescence were measured. After irradiation, cell death ratio was calculated and morphological changes in the cells were observed. The results showed that the content and photodynamic effects of cellular PpIX presented in the order Hep-2 cells > HaCat cells > fibroblasts, either co-cultured with ALA alone or with ALA plus DFO. DFO was found to have increasing effects on both PpIX level and cell death ratio in the same order. It was found that DFO had different potentials for augmenting ALA-PDT in these cutaneous cell lines. The cells proliferating more rapidly might be more susceptible to enhancement of DFO.