Transparent background in a Tkinter window

It is possible, but it's OS-dependent. This will work in Windows:

import Tkinter as tk # Python 2
import tkinter as tk # Python 3
root = tk.Tk()
# The image must be stored to Tk or it will be garbage collected.
root.image = tk.PhotoImage(file='startup.gif')
label = tk.Label(root, image=root.image, bg='white')
root.overrideredirect(True)
root.geometry("+250+250")
root.lift()
root.wm_attributes("-topmost", True)
root.wm_attributes("-disabled", True)
root.wm_attributes("-transparentcolor", "white")
label.pack()
label.mainloop()

Here is a solution for macOS:

import tkinter as tk

root = tk.Tk()
# Hide the root window drag bar and close button
root.overrideredirect(True)
# Make the root window always on top
root.wm_attributes("-topmost", True)
# Turn off the window shadow
root.wm_attributes("-transparent", True)
# Set the root window background color to a transparent color
root.config(bg='systemTransparent')

root.geometry("+300+300")

# Store the PhotoImage to prevent early garbage collection
root.image = tk.PhotoImage(file="photoshop-icon.gif")
# Display the image on a label
label = tk.Label(root, image=root.image)
# Set the label background color to a transparent color
label.config(bg='systemTransparent')
label.pack()

root.mainloop()

(tested on macOS Sierra 10.12.21)