Using dis to look at the bytecode generated for the two versions:
not ==
  4           0 LOAD_FAST                0 (foo)
              3 LOAD_FAST                1 (bar)
              6 COMPARE_OP               2 (==)
              9 UNARY_NOT           
             10 RETURN_VALUE   
!=
  4           0 LOAD_FAST                0 (foo)
              3 LOAD_FAST                1 (bar)
              6 COMPARE_OP               3 (!=)
              9 RETURN_VALUE   
 
The latter has fewer operations, and is therefore likely to be slightly more efficient.