Can you be more specific? The fall is three months long, and the temperature around the lake in the fall can high in the 50's in October, and in November it can be in the 40's, approaching freezing at night. There's still life there and a reason to visit, but a lot closes. That's another point. Lake Como is huge. Going to Lake Como, you usually choose a town on the lake to visit, not the lake itself. You visit Bellagio, Varenna, Menaggio, etc. You have to decide where in Lake Como, and since you are going in the fall, find out which hotels are open. the fall is September 22nd to December 20th this year. Places that will be open in September will be closed in December on the lake.

Twelve nights, with tired day of arrival, and packing night the evening before departure, really means 10 nights to visit and soak up Italy. Four places in 10 nights is quite a lot. Assisi is a ways from Florence. It's a full, not too convenient day trip, leave in the morning, get back late, assuming you are not driving. If you go to Assisi that would leave you 9 nights to really Venice, Florence, and the town you choose to stay at around the lake. That's only 3 days per place, averaged out. You wouldn't get to see much of Venice or Florence in 3 days.

It would help to be more specific about your time and itinerary.