Pereskia aculeata has been widely investigated due to its anti‐inflammatory potential. Among the metabolites found in this species are the phytosterols beta‐sitosterol (β‐SIT) and stigmasterol (STIG). The objective of the study was to evaluate the anti‐inflammatory and toxicity activities of the hexane partition of P. aculeata (PHEX), as well as β‐SIT and STIG. PHEX was prepared and the phytosterols were quantified. In terms of toxicity against L929 fibroblast cells, PHEX showed toxicity up to 200 µg/mL; STIG and β‐SIT showed toxicity up to 25 µg/mL. PHEX inhibited 66% of nitric oxide radicals, while STIG and β‐SIT inhibited 33.73% and 34.94%, respectively. In an anti‐inflammatory test against Zophobas morio larvae, all samples significantly reduced hemocyte levels. Additionally, the LD50 values were calculated: 229.6 mg/kg for PHEX, 101.5 mg/kg for STIG, and 103.8 mg/kg for β‐SIT. In conclusion, the study indicates that the phytosterols present in PHEX may contribute to its anti‐inflammatory activity.