You can create an new list with lowerCase characters and then to check with it.
current_users = ["John", "Admin", "Jack", "Ana", "Natalie"]
new_users = ["Pablo", "Donald", "Calvin", "Natalie", "Emma"]
current_users_lower = map(str.lower,current_users)
for username in new_users:
if username.lower() in current_users_lower:
print("Username unavailable.")
else:
print("Username available.")