Background: Despite the widespread ethnopharmacological use and significance of Silybum marianum (SM) seeds, most of its phytochemical and biological properties are still yet to be confirmed using validated scientific methods. Objectives: The current study was designed to investigate the phytochemical and biological properties of SM seed extract. Materials and Methods: Methanolic extract of SM (MESM) dried seeds was fractionated by column chromatography, and fractions (SA1–SA10) were evaluated for antioxidant, antimicrobial, and cytotoxic activities (brine shrimp lethality assay and antileishmanial assay). Results: All fractions showed considerable level of antioxidant potential. Free radical scavenging activity of fraction (SA9) was maximum at 80.7%. Fraction SA4 exhibited substantial total antioxidant capacity (101.81 μg/mg). In ferric-based reducing antioxidant power assay, fraction SA4 showed the highest antioxidant power (258.93 μg/mg). Phytochemical screening of the fractions (SA1–SA10) inferred that total phenolic contents were maximum in fraction SA7 (85.13 μg/mg) and total flavonoid contents were found to be highest in fraction SA1 (58.24 μgQE/mg). However, mild antibacterial and antifungal activities were shown by different fractions. To evaluate cytotoxic potential, brine shrimp lethality bioassay was performed. Among all the fractions, the fraction SA9 revealed the lowest LD50of 49.99 μg/mL, whereas all the other fractions tested demonstrated significant cytotoxic property. The results of antileishmanial assay showed that the fraction SA6 possesses the highest mortality percent (84%) compared to the other fractions. Conclusion: These findings revealed that MESM can be an important source of natural antileishmanial herb that can be used as a therapeutic alternative for leishmaniasis.