Infertility constitutes a global health dilemma, affecting approximately 10–15% of all couples. Although semen analysis constitutes the backbone of the diagnosis of male infertility, the contribution of a plethora of hormonal and nutritional factors cannot be ignored, particularly in the setting of normal semen analysis results. The current study sought to determine how serum levels of Testosterone, Estrogen, Prolactin, Thyroid Stimulating Hormone (TSH), free Thyroxine (fT4), folate, and homocysteine differed between male infertility patients and age-matched controls, as well as their effect on sperm parameters in infertile men. Seventy diagnosed cases of male infertility and 70 age-matched controls were recruited after acquiring informed consent from each subject. Blood samples were collected and analyzed for the above hormones and nutritional parameters by using a chemiluminescence-based automated platform. Serum levels of prolactin (P <0.0001), testosterone (P<0.0001), Estrogen (P<0.0001), TSH (P<0.0001) and homocysteine (P<0.0001) were signicantly higher, while serum levels of fT4 (P<0.0001) and Folate (P = 0.0015) were signicantly lower in infertile males vis-a-vis age-matched controls. Moreover, a signicant positive correlation of serum Estrogen levels with sperm count (r = 0.3300; p = 0.01) and a signicant negative correlation of serum Prolactin with sperm motility (r = -0.5017; p<0.0001) were observed in cases of male infertility. Besides routine semen analysis, various hormonal and nutritional factors can aid in cementing the diagnosis of male infertility. Summary: Ÿ Semen analysis constitutes the backbone of the diagnosis of male infertility. Ÿ However, the contribution of hormonal and nutritional factors cannot be ignored. Ÿ Blood samples were analyzed for hormones and nutritional parameters. Ÿ Various factors aid in cementing the diagnosis of infertility, besides routine semen analysis. Ÿ Correction of these factors may potentially help to improve fertility status in men. Ÿ Semen analysis constitutes the backbone of the diagnosis of male infertility. Ÿ However, the contribution of hormonal and nutritional factors cannot be ignored. Ÿ Blood samples were analyzed for hormones and nutritional parameters. Ÿ Various factors aid in cementing the diagnosis of infertility, besides routine semen analysis. Ÿ Correction of these factors may potentially help to improve fertility status in men.