Artemis, Baillie Gifford, Khazanah, Vitruvian Partners and Yahoo! Japan will acquire an undisclosed share of the Edinburgh-based company which was crowned business of the year at the Scottish Business Awards last year.
The new backers will join existing investment partners SEP and Sequoia, with officials hoping the funding will accelerate its growth in the £332bn worldwide online travel market.
Skyscanner chief executive, Gareth Williams, said: "Skyscanner has enjoyed high double-digit growth rates for some years now and has been profitable since 2009.
"This success is thanks to our 1,200 direct partner relationships, the trust of the 50 million travellers who use us every month, our technology and the dedication of our teams to deliver the best experience for travellers possible.
"This financing round and our recent new hires allow us to build fantastic further tools for travellers.
"I'd like to thank SEP and Sequoia for their belief in us and also welcome our new investors to the team. We have so much more to do together - it's an exciting time."
With the support of the new investors, Skyscanner will secure additional access to virtual and geographical marketplaces around the world, expanding their online user profile.
The travel website was officially launched in 2003 and has grown to become the number one flight search engine in Europe.
The site is used by more than 50 million people each month to book flights, hotels and car hire, with their free mobile app being downloaded over 40 million times.
Skyscanner now operates worldwide with offices in the UK, Singapore, Beijing, Shenzhen, Miami, Barcelona, Sofia and Budapest. Offering travel searches in more than 30 different languages.