Biostimulants represent a sustainable strategy to enhance the therapeutic potential of medicinal plants, which often exhibit low and variable levels of bioactive compounds. Cannabis sativa, a medicinally important species, produces diverse cannabinoids, such as THC, CBD, CBG, and CBC, whose profiles depend on plant chemotype and determine pharmacological activity. We developed a novel plant-based biostimulant, Tricostimulant™, to optimize cannabinoid production in Cannabis sativa. Field trials demonstrated increased biomass and selective enhancement of cannabinoid content. In high-CBD chemotypes, Tricostimulant™ was associated with higher CBD and CBG without relevant changes in THC levels, whereas in high-THC chemotypes, higher THC values were observed without evident variation in CBD. The most pronounced differences were observed when the biostimulant was applied during the vegetative stage, highlighting the importance of application timing. These results indicate the potential of Tricostimulant™ to modulate cannabinoid profiles, contributing to improved optimization and standardization of cannabis-based therapeutics. Further research is required to confirm these findings and elucidate the underlying mechanisms of biostimulant action.