The cactus family plant has been used in folk medicine for a long time. In this work, Opuntia stricta chemical composition and its antioxidative and anti-inflammatory properties were investigated. Our results showed that O. stricta is highly rich in fibers and minerals. The present study assessed the levels of polyphenol contents and antioxidant and in vivo anti-inflammatory activities. The highest phenolic compounds and antioxidant activity were observed in the methanolic extract. Concerning the qualitative analysis, nine phenolic and organic acids were identified and quantified by high-performance liquid chromatography (HPLC). Luteolin-7-Glu (4.25 μg/g), apigenin-7-Glu (3.15 μg/g), and catechin (2.85 μg/g) were identified as major phenolic compounds. The predominant fatty acids detected by gas chromatography (GC) coupled to a flame ionization detector were linoleic and linolenic acids (35.11%). A factorial design plan was used to determine the effect of temperature, agitation speed, and maceration period on phenolic contents. In vivo, the methanol extract from Opuntia stricta showed anti-inflammatory activity. The computational modeling reveals that O. stricta compounds bind VEGF, IL-6, and TNF-α with high binding scores that reach −8.7 kcal/mol and establish significant molecular interactions with some key residues that satisfactorily explain both in vitro and in vivo findings. These data indicate that Opuntia stricta cladode powder could be potentially useful in pharmaceutical and food applications.