Gloucestershire handed the Unicorns a second heavy defeat in a row in Group C of the Yorkshire Bank 40.
The Unicorns, a team made up of part-time cricketers looking to get back into the professional game, went down by nine wickets after mustering 153-9 with the bat at Wormsley.
Gloucestershire knocked off their target in 23.2 overs with openers Hamish Marshall and Michael Klinger leading the charge.
Marshall struck eight fours and a six in his 62-ball 67 before being caught by Garry Park off left-arm spinner Luke Beaven (1-34) to end a first-wicket stand of 111.
Gloucestershire skipper Klinger saw the job through to its conclusion, finishing on 52 not out alongside Chris Dent (29no).
Earlier, David Payne claimed 3-29 as the Unicorns - who lost by eight wickets to Somerset in their Group C opener the previous day - struggled after choosing to bat first.
Ex-Glamorgan batsman Michael O'Shea's 41 was the top score, while Payne received good support from fellow seamer Will Gidman (2-21) and left-arm spinner Ed Young (2-31).