The current work is focused on establishing therapeutic protocol using unconventional drugs of herbal origin and studying their mechanism of action at molecular level in the treatment of bovine sub-clinical mastitis. It explores the potential of different cytokines which can be used for diagnosis, prognosis and monitoring of bovine sub-clinical mastitis. alkaloids was administered intramammarily in 24 sub-clinically affected quarters once a day for 5 consecutive days at the rate of 10 ml of 1% formulation. In 18 disease control quarters, sterile normal saline was infused. The bacterial cultural examination, somatic cell count (SCC) and cytokines (IL-1β, IL-6, IL-8, IL-12, GM-CSF, IFN-γ, TNF-α) expression by real-time PCR were evaluated on day 7, 14, 21 and 28 post-last treatment from milk samples. Around 75.0% of treatment group quarters showed significant ( < 0.05) reduction in SCC on day 28 post-last treatment, whereas 94.4% control group quarters did not show any significant decline in SCC. 58.3% of treated quarters showed both bacteriological cure as well as significant ( < 0.05) reduction in SCC on day 28 post-last treatment. While, among control group quarters, 83.3% quarters not only remained bacteriological positive, they also did not show any significant decline in SCC. The in vitro antimicrobial activity of alkaloids of was evaluated. Lower concentrations of alkaloids (0.25% and 0.50%) dissolved in normal saline showed zone of inhibition against 12 out of 15 isolates, however higher concentration (1, 1.5, 2, 2.5 and 5%) showed zone of inhibition against all 15 bacterial isolates. The gene expression level of IL-1β, IL-8 and IFN-γ cytokines exhibited significant difference between healthy and sub-clinically affected quarters highlighting the potential of these cytokines in the diagnosis of bovine sub-clinical mastitis. Down-regulation of IL-1, IL-6, IL-8 and IFN-γ cytokines in treated quarters can be explored for making the prognosis and monitoring post-treatment disease progression of bovine sub-clinical mastitis. The alkaloid demonstrated strong in vitro and in vivo antibacterial activity, along with causing immunomodulation by enhancing post-treatment gene expression of IL-1, IL-6 and IL-8 cytokines. Therefore, alkaloids hold a strong claim as an effective alternative herbal therapy in bovine sub-clinical mastitis.