Ocimum gratissimum is a shrub that belongs to the Lamiaceae family of plants. Despite the known biological activities and ethnomedicinal applications, comparative evaluation of the effects of different extraction techniques on the chemical and bioactive properties of O. gratissimum extracts has not yet been performed. This study adopted different analytical techniques to determine the effect of extraction temperature and solvent type on the phytochemical and bioactive properties of O. gratissimum extracts. Chemical profiling showed increased concentrations of compounds for both the ethanolic and methanolic extracts compared to the water extracts. The results also revealed that the extraction temperature had an effect on the total phenolic content and radical-scavenging properties of the different extracts. The antioxidant kinetic modeling achieved the best fit when using the second-order kinetic model. Methanolic extracts had the highest levels of antibacterial activity against Escherichia coli, Bacillus cereus, Staphylococcus aureus, and Salmonella typhimurium. At high concentrations, all extracts lowered the viability of the breast cancer cell line MDA-MB-231. In conclusion, the chemical and bioactive properties of all extracts showed significant dependence on the extraction temperature and solvent type. With proper extraction methods, they boast a wide range of promising applications in the medical, pharmaceutical, and food industries.