Introduction: Toxoplasmosis remains globally relevant because of its consequences on pregnancy outcomes, mental disorders, and opportunistic tendencies in immunocompromised individuals. Current medications have severe side effects on the host, low efficacy against the parasites, and potential development of resistance. This emphasizes the need to discover better and safer drugs to treat toxoplasmosis. This study evaluated the in vitro effects of ethanolic leaf extracts of Andrographis paniculata (EEAP) on protein kinase G (PKG) involved in cell invasion by T. gondii. Methods: EEAP was obtained through the maceration of dried leaf powder and then subjected to qualitative and quantitative screening of phytochemicals. Vero cells infected with the RH strain of T. gondii were used to evaluate the cytotoxicity and antiparasitic potential of EEAP, andrographolide, and clindamycin using the MTT assay. Microscopy was used to determine the cell invasion and intracellular replication of tachyzoites in the treated infected Vero cells at 24 h using 4 h post-infection treatment models. Gene expression profiling of TgPKG was performed using RT-qPCR. The expression of microneme proteins was determined using immunoblotting. Results: EEAP was found to contain more terpenoids. EEAP, andrographolide, and clindamycin were safe for host cells. EEAP and andrographolide exhibited good anti-parasitic activities against T. gondii. Microscopic assessment revealed a low infection index and intracellular replication by EEAP and andrographolide. RT-qPCR revealed a significant (P=0.002) downregulation of the TgPKG gene by 0.114-fold (88.6% decrease) and 0.068-fold (93.2% decrease), respectively, compared with the control. The expression of TgMIC1(0.442-fold, 55.8% decrease) and TgMIC2 (0.385-fold, 61.5% decrease) proteins significantly (P= 0.0001) decreased compared to the control. Conclusion: This study showed that EEAP and andrographolide are promising drug candidates effective against T. gondii, safe for the host cells, and can be used for the development of a potent anti-Toxoplasma drug to target the TgPKG gene involved in cell invasion to prevent disease progression.