This study aims to assess for the first time the content of phytosterols (PS) in espresso coffee (EC) to deepen the knowledge about the phytochemicals and health potentials of coffee brews. PS were extracted by hot saponification from 14 EC samples produced with coffee originating from 13 coffee-producing countries. PS were identified and quantified by high-performance liquid chromatography (HPLC) after derivatization. Among the detected PS, β-sitosterol (4.1–18.2 mg/L) was the most abundant followed by stigmasterol (1.1–4.9 mg/L), campesterol (0.9–4.7 mg/L), and cycloartenol (0.3–2.0 mg/L). Total PS fraction ranged from 6.5 mg/L to 30.0 mg/L with an average level of 15.7 ± 5.8 mg/L. Therefore, a standard cup of EC (25 mL) could provide 0.4 ± 0.1 mg of PS.