The purpose of this study is to evaluate the impact of multifunctional microbial fertilizer and inorganic macronutrient fertilizers N, P, K on plant biomass, essential oil yield, chemical composition, antimicrobial and antioxidant activities of tulsi (Ocimum tenuiflorum L.) grown in Hanoi, Vietnam. Among the four formulas tested, F2 emerged as the most favourable, comprising a basal fertilization of 10 tons of decomposed manure, 15 kg of multifunctional microbial fertilizer, 150 kg of P fertilizer, and 75 kg of K fertilizer, together with top dressing of 200 kg of N fertilizer and 75 kg of K fertilizer per hectare. This formula yielded the highest tulsi dry biomass (4.73 ton/ha), essential oil yield (0.831%), and essential oil production (39.29 L/ha), which were significantly different (p < 0.001) from those of the remaining formulas. In addition, the concentration of the essential oil main compound - eugenol (63.1%) of tulsi in F2 was also the highest, which may be the cause of the strongest antibacterial activity against Bacillus subtilis, Escherichia coli (IC50 of 320, 273 µg/mL, respectively), and antifungal activity against Candida albicans (IC50 of 400 µg/mL), as well as antioxidant activity (IC50 of 0.165 µg/mL) compared to the other formulas tested. The results suggested the role of both multifunctional microbial fertilizer and inorganic macronutrient fertilizers N, P, K on growth and essential oil biosynthesis in tulsi. These findings are important in identifying the most efficient fertilization formula for cultivating tulsi, serving the needs of the cosmetic and medicinal industries.