Staphylococcus sp. as Gram-positive and Escherichia coli as Gram-negative are bacterial pathogens and can cause primary bloodstream infections and food poisoning. Coagulation, flocculation, and sedimentation processes could be a reliable treatment for bacterial removal because suspended, colloidal, and soluble particles can be removed. Chemical coagulants, such as alum, are commonly used. However, these chemical coagulants are not environmentally friendly. This present study evaluated the effectiveness of coagulation, flocculation, and sedimentation processes for removing Staphylococcus sp. and E. coli using diatomite with standard jar test equipment at different pH values. Staphylococcus sp. demonstrated 85.61% and 77.23% significant removal in diatomite and alum, respectively, at pH 5. At pH 7, the removal efficiency decreased to 79.41% and 64.13% for Staphylococcus sp. and E. coli, respectively. At pH 9, there was a decrease in Staphylococcus sp. after adding diatomite or alum compared with that of E. coli. The different removal efficiencies of the Gram-positive and Gram-negative bacteria could be owing to the membrane composition and different structures in the bacteria. This study indicates that diatomite has higher efficiency in removing bacteria at pH 5 and can be considered as a potential coagulant to replace alum for removing bacteria by the coagulation process.