On my way to Munich from Brussels I have one day to spend in either Berlin or Koln before catching an overnight train to Munich. Which city is most worth seeing?

Berlin, without a doubt. The Köln cathedral is magnificent, and there are various museums and churches, but in my opinion, it doesn't hold a candle to Berlin.

Thanks Pegontheroad! Any suggestions on what I should definitely see while there?

Berlin is much more interesting but you need 3-4 days - it's just huge. I would stop in Cologne, see the cathedral and a museum that interests you, then take the train south to Braubach and tour Marksburg Castle - the only never-destroyed castle on the Rhine - before heading on to Munich. This is a much more do-able trip and involves some very scenic train travel along the Rhine.

If you are interested in modern architecture, ancient and modern art collections, vibrant city scene and music, Berkin is the place to go. On the other hand, the Cathedral, and restored romanesque churches in Koln are very impressive also, besides several excellent museums.

From Brussels to Munich, Berlin is a huge detour while Köln is on the way. Just a thought.

Half a day - no more than half a day because you'll spend the whole first half of that day on the train to get to Berlin - is nothing for a city like Berlin. Regarding the short amount of time you have, I'd vote for Köln in an instant.

If you've never been to Germany before, could you re-arrange the schedule to take a day train from Koln to Munich instead of the night train? It runs through some scenic areas. It's not THAT long a ride...under 5 hours from Cologne. For example, you could catch the earliest train from Brussels, spend the day in Cologne, overnight in the train station Ibis hotel, then catch a morning train to Munich (1 change).
Or catch an early (around 7 am) from Brussels to Cologne, arrive just after 9 am, sightsee for several hours and catch a late afternoon train to Munich. If you're traveling in summer months, you could catch a train in Cologne around 3:30 pm and still be in Munich before dark. Plenty of luggage storage facilities at Cologne.

Berlin would be fine if your one day doesn't include the time you need to travel from Brussels to Berlin. But it is really out of the way and simply not on the route between Brussels and Munich.

Fully agree with quokka. A detour via Berlin means a very long and boring train ride. Certainly not worth for a single day.

I've been to both many times and I totally agree with BTilke's recommendations.

Berlin is much larger and has a lot more to see and do and to only spend one day there would be unfortunate IMO and, as has been stated, out of the way.

You can easily store your luggage in the automated storage facility at the Cologne station and the cathedral is literally right outside the station door.

If you are more interested in speed than sightseeing along the Rhine, book yourself onto a train from Cologne southbound which makes use of the "high speed" segment between Cologne and Frankfort and takes only 57 minutes.

I'd do Koln. It's a great city and very seeable in a day. You could also spend the morning in Bonn and afternoon/evening in Koln.

Berlin is a long way out of the way (much more time lost to travel) and it is really a place that needs at least 3 or 4 days rather than one.

Absolutely, Cologne is on the way and 'can be seen' in a day. It also has great nightlife if you're so inclined.