Cannabis sativa L. is increasingly recognized for its therapeutic potential beyond its psychoactive properties and use in fiber production. Recent research highlights its efficacy in treating pain, anxiety, inflammation, and as an antioxidant. These benefits are attributed to a variety of bioactive compounds, including cannabinoids, terpenes, and flavonoids, which have sparked interest in their medicinal applications. This study investigated the phytochemical composition and biological activities of two C. sativa strains, with a particular focus on identifying functional groups using FTIR. Crude extracts from C. sativa buds were prepared using solvents of varying polarities. Phytochemical contents were quantified using standard techniques. Antioxidant activity was measured via TLC, DPPH, and FRAP assays, while antimicrobial activity was assessed using the agar well diffusion method. FTIR analysis was used to detect functional groups in the extracts. Phytochemical analysis showed TPC values ranging from 11.77 to 93.51 mg GAE/g and TFC values from 25.61 to 45.95 mg QE/g. The ethyl acetate extract (high THC) exhibited the highest TPC (93.5 mg GAE/g) and a DPPH IC50 of 0.0065 µg/L, indicating strong antioxidant activity. Cheese 2 extracts (ethanolic and n-hexane) showed higher TFC values (45.95 & 45.59 mg QE/g). TLC confirmed antioxidant activity. The DPPH assay confirmed strong radical scavenging activity, while FRAP displayed notable activities in neutralizing free radicals. Both strains displayed notable antimicrobial activity against E. coli, S. aureus, B. cereus, and S. typhi. FTIR analysis identified key functional groups related to cannabinoids, terpenes, and fatty acids. These findings highlight C. sativa as a promising source of bioactive compounds with antioxidant and antimicrobial properties. However, further research is needed to fully assess its antioxidant potential.