Buses still account for three-quarters of public transport journeys, but passenger numbers fell to 409 million in 2015-16 compared to the most recent peak of 487m in 2007-08.
This includes the total went down by 5m since the last annual figures were published.
Pat Rafferty, Scottish secretary of the union, which is campaigning for buses to be returned to public control, said: “These numbers show the Scottish Government is failing bus passengers.
“Without bus regulation, we will continue to have a free-for-all where the only thing that matters is profit – with people and passengers just being the mugs who pick up the tab.
“Scotland’s bus services are in a mess. The public are paying more and more both in fares and in the amount of subsidy they are giving to the bus companies.
"But the service they are getting continues to become worse and worse.”
Friends of the Earth Scotland air pollution campaigner Emilia Hanna said: “The bus sector in Scotland is in dire straits, with only Edinburgh bucking the trend of decline.
“Rural services are being cut, urban services are often confused and don’t link up well as a network, and fares continue to increase at the expense of those who depend on the bus.
“Buses are a big part of the solution to air pollution and the Scottish Government urgently needs to reform how buses are regulated to make it easier for local authorities to operate functioning bus networks which work in the public interest rather than try to join up piecemeal services.”
Colin Howden, director of sustainable transport campaigners Transform Scotland, said: “It’s very worrying to see this year-on-year decline in bus use.
“Buses play an important role in reducing congestion in urban areas and providing services to lower-income groups who often don’t have access to cars.
“It’s deeply disappointing the Scottish Government continues to cut investment in bus services - government funding has reduced by 5 per cent over the past five years, and this week’s Parliamentary debate on the Scottish Budget looks set to agree a further 3 per cent cut in bus investment over the next year.
“The Government should instead be taking action to cut congestion by putting in place bus priority in urban areas, and protecting threatened rural bus services.”
Liberal Democrat transport spokesman Mike Rumbles said: “The number of people using buses has dropped dramatically, as has the number of buses and journeys available to people.
“That means more traffic on our roads, less investment in public transport and missed targets on climate change.”
A spokesman for the Confederation of Passenger Transport, which represents bus operators, said: “Several factors have impacted upon bus patronage over the past decade, including a double-dip recession, low costs of car ownership, changing social patterns which have led to more internet shopping and less high street retail footfall, and the ever-growing problem of congestion.
"Mr Rumbles is right to highlight the need for proper investment in sustainable and active transport.
"Local and national government can further help drive demand in bus use by working in partnership with the industry to give buses – and bus passengers – priority through traffic.
"The bus industry in Scotland has invested hundreds of millions over the last ten years in improvements such as low emission buses, smart-ticketing and real-time information, but if bus speeds remain compromised by growing car use it will remain a difficult task to generate modal shift to bus.”
A spokeswoman for the Scottish Government's Transport Scotland agency said: “The Scottish Government is committed to improving bus services and the workings of the current regulatory framework through partnerships with bus operators and local transport authorities.
"We are concerned about the decline in bus patronage, something which has been continuing since at least the 1960s, well before de-regulation began in the mid-1980s.
"However, it is important to note the decline is not seen in all areas.
"Indeed, some areas of Scotland have shown growth over the last five years, whereas others see double figures in terms of percentage of decline.
“That is why the solution must be local.
"This Government will bring forward a transport bill that will give local authorities the framework to work in partnership with bus operators to improve services.
"Whilst we have no plans for wholesale re-regulation, we do want to see more people using our public transport networks.
“We continue to spend nearly a quarter of a billion pounds a year in grants to support the network, promote the take up of new greener buses and reimburse operators for free bus travel provided to older and disabled people under the national concessionary travel scheme.
"We are also investing over £1 billion per year in public and sustainable transport to encourage people onto public transport and active travel modes.”