Background: Lasers are widely used in medicine in soft and hard tissue surgeries and biostimulation. Studies found in literature typically compare the effects of single-wavelength lasers on tissues or cell cultures. In our study, we used a diode laser capable of emitting three components of visible light (640 nm, red; 520 nm, green; 450 nm, blue) and combining them in a single beam. The aim of the study was to assess the effects of laser radiation in the visible spectrum on tissue in vitro, depending on the wavelength and pulse width. Methods: All irradiations were performed using the same output power (1.5 W). We used various duty cycles: 10, 50, 80 and 100% with 100 Hz frequency. Maximum superficial temperature, rate of temperature increase and lesion depth were investigated. Results: Maximum superficial temperature was observed for 450 + 520 nm irradiation (100% duty cycle). The highest rate of increase of temperature was noted for 450 + 520 nm (100% duty cycle). Maximum lesion depth was observed in case of three-wavelength irradiation (450 + 520 + 640 nm) for 100, 80 and 50% duty cycles. Conclusions: The synergistic effect of two-wavelength (450 + 520 nm) irradiation was observed in case of maximum temperature measurement. The deepest depth of lesion was noted after three-wavelength irradiation (450 + 520 + 640 nm).