Photobiomodulation (PBM) has been applied to manipulate cellular responses by using monochromatic light in different wavelengths from ultraviolet (UV) to infrared (IR) region. Until now, an effective wavelength has not been revealed to induce proliferation and/or differentiation of cells. Therefore, in the presented study, we decided to use a specially designed plasma arc light source providing wavelengths between 590 and 1500 nm in order to investigate its biomodulatory effects on chitosan scaffold-supported three-dimensional (3D) cell cultures. For comparison, two-dimensional (2D) cell cultures were also carried out in tissue-culture polystyrene dishes (TCPS). The results showed that light-induced temperature rise did not affect cells when the distance between the light source and the cells was 10 cm and the frequency of administration was daily. Moreover, light was applied for 5 and 10 min to the cells in TCPS and in chitosan scaffold groups, respectively. Cell culture studies under static conditions indicated that polychromatic light significantly stimulated bone nodule formation via the prolonged cell survival and stimulated differentiation of MC3T3-E1 preosteoblastic cells in both TCPS and chitosan scaffold groups. In conclusion, specially designed plasma arc light source used in this study induces formation of bone tissue and so, this light source is proposed as an appropriate system for in vitro bone tissue engineering applications. Statistical analyses were performed with one-way ANOVA by using GraphPad Instat software and standard deviations were calculated by using data of three parallel samples for each group.