The company covered 712 miles around Scotland as they performed their latest show, Tosca.
As reported in the Evening Times the performers were inspired to shun the tour bus for the sponsored trek by their colleague, opera singer Jenny Wilson-Best.
The 52-year-old, from the West End, has neurological condition multiple sclerosis (MS) and is starting to lose her mobility.
The cash will go to charity Revive MS Support, which has a base in Maryhill.
Scotland has the highest incidence of the disease, which Ozzy and Sharon Osbourne's son Jack, 26, has just been diagnosed with.
Jenny joined in parts of the walk, and also sang after each leg, which was done in a relay.
She said: "We met a lot of people who said things like 'my aunt has MS and we don't know about Revive' so the great thing was that we were really able to say about this wonderful centre.
"On the walk, one 92-year-old pensioner gave one of the girls £20, and one of the walkers came up to me and took my hand and said: 'I'm doing this walk because I know that I'm lucky enough that I can'. It was very touching."
All the opera firm's departments took part in the walk, from the orchestra, to the wardrobe and wigs department.
It was organised by chorus members Bronagh Byrne and Lois Johnston.
Bronagh, 38, who has worked with Scottish Opera for six years, said: "It was just an amazing experience.
"It's changed a lot of people's lives, not just about MS awareness, but lots of people are now thinking of joining walking clubs."
The walk began in Glasgow on May 13, and walkers paced to Inverness, then back to Edinburgh before heading to Aberdeen and then back to Glasgow.
Cara Doran, fundraising manager for the charity, said: "We've been overwhelmed by Jenny's enthusiasm to do something like this and the generosity of all her friends to give up so much of their time and energy."
Jenny, who is from Chichester, Sussex, has had a 30-year opera career and also worked for the English National Opera and Paris-based Opera company Les Arts Florissant.
To make a donation, visit www.justgiving.com/jennywilsonbest.