Commercial polystyrene (PS) cell culture flasks were employed as containers of target brain tumor cells exposed to ultrasound. However, a problem was observed in that ultrasound energy was reflected at the surface of the flask with a different specific acoustic impedance from the water outside of the flask and the culture medium in the flask, and did not reach the cells sufficiently. Therefore, we prepared the original cell culture flask. This cell culture flask has thin polymer film acoustic windows for ultrasound irradiation at the top and bottom ends of a hollow cylindrical plastic pipe. We reached the conclusion that it is desirable to weld the poly(ethylene terephthalate) (PET) film or PS film as an acoustic window to the flask body by heat welding. The PS film acoustic window could be welded to the end of the PS or poly(menthyl methacrylate) (PMMA) flask body, but PET film acoustic window could not be welded to the end of the PS or PMMA flask body. The PET film acoustic window welded to the flask main body by ultrasound welding has sufficient durability to ultrasound sonication and excellent cellular adhesiveness.