The minimal invasiveness and high selectivity of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT; 5-ALA-PDT) renders it a viable therapeutic option for the treatment of various types of cancer. Compared with conventional lasers, light-emitting diodes (LEDs) are an inexpensive and convenient low-energy light source option. Nevertheless, the scope of LEDs in the 5-ALA-PDT of colorectal cancer (CRC) has yet to be fully determined. Thus, the aim of the present study was to assess the efficacy of LEDs in the 5-ALA-PDT of colon cancer in vitro by evaluating cytotoxic activity. 5-ALA-treated human CRC cells (SW480) were irradiated with LEDs of varying wavelengths: Red (630 nm), green (515 nm), blue (456 nm) and violet (399 nm). An MTS assay was conducted to determine cell viability. Additionally, a concentration-response experiment was conducted with the most therapeutic wavelength (violet) to examine the 5-ALA pharmacodynamics in vitro. The results revealed that only violet light in 5-ALA-PDT produced antitumour activity; this combination alone produced a drug concentration-and energy-related decrease in cell viability. The decrease in viability was partially reversed by 3-methyladenine, but not by Z-VAD(OMe)-FMK, suggesting that 5-ALA induced the autophagy, but not the apoptosis of SW480 cells. The nature of the multi-well plates used markedly affected the effectiveness of PDT. Black-walled plates appeared to protect approximately 25% of cells from the effects of PDT. By contrast, clear plates permitted light access to the wells, even when protected from direct PDT treatment. On the whole, the findings of the present study indicate that the use of LEDs in 5-ALA-PDT in vitro induce the fluence-dependent tumour cell death of SW480 cells. The choice of multi-well plates greatly affects the results obtained in vitro. The antitumour effect was high with violet light. Hence, the use of LEDs in 5-ALA-PDT may prove to be an effective potential treatment for CRC.