Precise crop fertilization requires an in-depth understanding of plant uptake and utilisation to optimise sustainable production. This study investigated the influence of nitrogen (N) nutrition and pruning on the cannabinoid concentrations and biomass of a commercial cannabis cultivar; the rationale for this study is how N supply and pruning affect cannabinoid yields and concentration in a commercial setting. Clones of a Cannabis sativa L. (CBD-type) were grown in a controlled-environment glasshouse in pots with coarse sand. After five weeks of vegetative growth under 210 mg/L N and an 18 h light regime, rates of 30, 60, 210, and 500 mg/L N were applied to plants for twelve weeks and a light regime set at 12 h. Double stem pruning was applied as an additional treatment to investigate efficacy on biomass increase. Biomass, N concentrations, and cannabinoid concentrations were measured after the final harvest. Pruning treatment did not increase cannabinoid concentrations or affect biomass. It was coincidentally found that plants on the glasshouse edge with higher exposure to sunlight developed more biomass and higher cannabinoid concentrations. Only biomass in leaves was increased significantly via higher nitrogen nutrition. Cannabinoid concentration, as well as cannabinoid yield per plant were decreased with the increase in N supply. High rates of fertilizer are not recommended because of reduced cannabinoid concentration and biomass yield: the ideal N supply is likely to be between 60 and 210 mg/L. This research will benefit growers and advisors in understanding the complexity of effects of nitrogen fertiliser and pruning practices on plant biomass and secondary metabolite production in medicinal cannabis.