Ocimum sanctum Linn. (Family: Lamiaceae), commonly known as ‘Tulsi’ is used as a traditional medicine in India due to its versatile therapeutic properties, which contain certain secondary metabolites affecting physiological and biochemical process in humans. In the present study, we have investigated the effect of Ocimum sanctum on the fertility of albino mice. The oral dosage administration of the leaves extract of Ocimum sanctum in male mice was monitored for 15 days to 30 days. The sperm motility was evaluated with sperm counting using a hemocytometer and the presence of different biomolecules that is secondary metabolites and other herbal compounds in the leaf extract of OS was analyzed using Quadrupole Time of Flight Mass Spectroscopy (QTOF). The phytochemical screening depicted presence of alkaloids, flavonoids and saponins in the leaf extract of the plant. The antifertility effect of the plant might be due to the chemical compounds such as apigenin, luteolin, hopanes, galangin, pyrrolidine and tryptohypol present in the extract. The results reported the progressive decline in the sperm count, sperm motility, whereas a significant increase in sperm abnormalities was observed in case of the test male Albino mice. This indicated that the secondary metabolites present in the plant shows the negative effect on the process of spermatogenesis in mammals. Thus, the leaves of Ocimum sanctum confirmed as an effective anti-fertility agent in the albino mice.