In recent years, light emitting diodes (LEDs), due to their low energy consumption, low heat emission and specific wavelength irradiation, have become an alternative to fluorescent lamps (FLs) in plant tissue culture. The aim of this study was to investigate the effects of various LED light sources on the in vitro growth and rooting of plum rootstock Saint Julien (Prunus domesticasubsp.insititia). The test plantlets were cultivated under a Philips GreenPower LEDs research module illumination system with four spectral regions: white (W), red (R), blue (B) and mixed (W:R:B:far-red = 1:1:1:1). The control plantlets were cultivated under fluorescent lamps (FL) and the photosynthetic photon flux density (PPFD) of all treatments was set at 87 ± 7.5 μmol m−2s−1. The effect of light source on the selected physiological, biochemical and growth parameters of plantlets was monitored. Additionally, microscopic observations of leaf anatomy, leaf morphometric parameters and stomata characteristics were carried out. The results showed that the multiplication index (MI) varied from 8.3 (B) to 16.3 (R). The MI of plantlets grown under mixed light (WBR) was 9, lower compared to the control (FL) and white light (W), being 12.7 and 10.7, respectively. In addition, a mixed light (WBR) favored plantlets’ stem growth and biomass accumulation at the multiplication stage. Considering these three indicators, we could conclude that under the mixed light, the microplants were of better quality and therefore mixed light (WBR) was more suitable during the multiplication phase. A reduction in both net photosynthesis rate and stomatal conductance in the leaves of plants grown under B were observed. The quantum yield (Yield = FV/FM), which represents the potential photochemical activity of PS II, ranged from 0.805 to 0.831 and corresponded to the typical photochemical activity (0.750–0.830) in the leaves of unstressed healthy plants. The red light had a beneficial effect on the rooting of plum plants; the rooting was over 98%, significantly higher than for the control (FL, 68%) and the mixed light (WBR, 19%). In conclusion, the mixed light (WBR) turned out to be the best choice during the multiplication phase and the red LED light was more suitable during the rooting stage.