Abstract The quality and quantity of light are important factors in controlling in vitro plant growth in photoautotrophic systems. The aim of this study was to evaluate the influence of light quality (fluorescent, white, red, blue, red/blue, and distant red) on microstumps of a Eucalyptus urophylla clone in an in vitro photoautotrophic system, as well as the intensity of fluorescent light (60, 85, 100, and 140 μmol m–2 s–1) in the growth and production of microcutting. The number of shoots and microcutting, the size of the largest shoot, the stomatal density, chlorophyll, and carotenoid content were analyzed. Light quality altered plant growth, and fluorescent light intensity did not affect the microstumps’ production during the evaluation period. In white light-emitting diode (LED) light, there was higher production of carotenoids, with a lower initial production of microcuttings. A smaller number of shoots were obtained in blue LED. In general, the different qualities and light intensities tested allowed for the growth of the Eucalyptus urophylla clone grown in vitro, making it possible to obtain microcuttings under photoautotrophic cultivation. Study Implications In vitro propagation is a stressful process for plants and has limitations for commercial-scale Eucalyptus production. Fluorescent lamps, closed containers, and high sucrose concentrations are traditionally used. To reduce costs and improve production, the use of efficient light sources and photoautotrophic cultivation systems become alternatives. This study investigated the influence of light on the in vitro growth of a Eucalyptus clone in a photoautotrophic system. The quality was more important than the intensity of light. Foresters will be able to indicate the use of LEDs (light-emitting diodes) as a replacement for fluorescent lamps. This approach is useful in enhancing micropropagation techniques.