Precise and efficient control of supplemental lighting is vital to minimize electrical energy costs in controlled environment agriculture. Even though various environmental factors such as temperature, vapor pressure deficit, CO2 concentrations, and water status influence photosynthetic capacity, current supplemental light control strategies are controlled only based on ambient sunlight conditions. Meanwhile, chlorophyll fluorescence is widely used as an indicator of environmental stress and photosynthetic capacity on account of its easy and non-invasive measurement. A chlorophyll fluorescence-based biofeedback system has been proposed as an innovative approach for precise control of supplemental LED light intensities. The biofeedback system can dynamically optimize LED light intensities based on real-time measurements of chlorophyll fluorescence while allowing plants to decide the amount of supplemental light they need. The biofeedback system has been previously validated in a growth chamber, but its application in an actual greenhouse condition remains unexplored. The objective of this research was to implement the biofeedback system in a greenhouse environment for real-time control of supplemental light intensities based on photosynthetic activity. Additionally, the productivity and energy efficiency of the biofeedback strategy were evaluated and compared to conventional light control strategies. Two fluorometers (MINI-PAM; Heinz Walz, Effeltrich, Germany) were used to monitor the electron transport rate (ETR) and quantum yield of photosystem II (ΦPSII) every 10 minutes, and the Biofeedback system adjusted supplemental LED light intensities until the predefined target ETR and ΦPSII were achieved. Three popular greenhouse crops [lettuce (Lactuca sativa), sweet basil (Ocimum basilicum), and spinach (Spinacia oleracea L.)] were grown under five supplemental light conditions. Specific targets of 1) electron transport rate (ETR), 2) quantum yield of photosystem II (ΦPSII), 3) photosynthetic photon flux density (PPFD), 4) daily light integral (DLI), and 5) no control (ambient sunlight) were used to control supplemental light intensities. In contrast to conventional lighting control methods, the biofeedback system tailored supplemental light intensities according to not only sunlight levels but also temperature and humidity. The result underlines the effectiveness and energy efficiency of the biofeedback system that could integrate variable environmental factors in the greenhouse and apply them to adjust supplemental light intensities precisely.