A self-developed hydrodynamic cavitation device with combinations of circular multi-orifice plates with Venturi tubes from the Hydraulics Laboratory at Zhejiang University of Technology was used to kill pathogenic microorganisms in raw water. Its effect was manifested by detecting the killing rate of E. coli and total colony count. The killing rates of E.coli and total colony count under the different working conditions were detected by plate counting method. The effects of cavitation number, orifice arrangement, orifice number, cavitation time, raw water percentage, throat length on the killing rates of pathogenic microorganism were analyzed. The experimental results showed that increasing orifice velocity and orifice number, prolonging cavitation time, lowering cavitation number, increasing the number of orifices, improving orifices arrangement and choosing appropriate raw water percentage and extending the throat length, could increase the killing rates of E.coli and total colony count. Hydrodynamic cavitation has potential application prospect as a new technology with no disinfection by-products, safe and high efficiency.