Disruptions caused by extreme weather to the region's road and rail network are said to have cost Westcountry businesses £167 million.
A new survey by the South West Chambers of Commerce found that 65% of businesses in the region were affected by road closures and rail services delays and cancellations as a result of snow and flooding over the last few months.
Two respondents said disruptions had cost their businesses more than £500,000, with one saying that none of their employees had been able to get to work at one point because of problems on the road network.
In December, a landslide caused by severe flooding closed the rail line between Exeter and Tiverton for five days with train operator First Great Western advising passengers not to travel by rail to Devon and Cornwall.
At one point, a section of the A39 at Perranarworthal was also closed because of the floods, with dozens more minor roads across the region also affected and parts of the Somerset Levels under water up to three weeks later.
More than 350 businesses took part in the survey and the results are set to inform a lobbying campaign aimed at securing investment to upgrade the region's fragile road and rail infrastructure.
Derek Phillips, chairman of South West Chambers of Commerce, said: "Our message to Government is that the South West has received far less than its fair share of investment in transport infrastructure for far too long and it is time that this unacceptable situation ceases. We are specifically looking for electrification of the rail line south of Bristol into Cornwall, the dualling of the A303 and A30 along their entire lengths and much improved mobile and Wi-Fi connectivity along the arterial routes to the South West."
The Chambers are highlighting new statistics which show that the average public sector spend for transport in 2010/11 was just £212 per person in the South West, the lowest figure in the UK, and just over a quarter of the spend per person in London.
They say that transport problems have meant the late or non-arrival of staff and clients; cancelled or postponed appointments and business trips; cancelled hotel bookings; a drop in customers for retail companies and resource issues for businesses relying on an inward supply of goods.
This resulted in lost revenue, loss of working days, reduced productivity and additional costs.
The survey found that 25% of businesses secure more than 75% of their revenue from outside the South West, and a third of South West businesses use transport links to get to and from other parts of the UK on a daily basis, with a further third using them weekly.
David Parlby, chief executive of Plymouth Chamber of Commerce, said: "On-going transport connectivity issues are more than just an inconvenience for the South West. The results of the survey show that businesses in the region are literally paying the price for the poor infrastructure and lack of investment.
"This is a critical issue which impacts on South West businesses of all sizes and from all industries, every day. Appropriate levels of investment in our transport connectivity are fundamental to the region's future economic development and ability to attract new businesses and inward investment."
David Bayliss, divisional director at the Plymouth office of architects Stride Treglown, said his business had been affected as staff were delayed as they tried to attend meetings at its head office in Bristol.
"In terms of getting people down here for meetings and for us to get to meetings, it does take a lot out of your day using the transport system," he said. "We only have one road in and one rail line in, there aren't alternative routes so if there is a problem, it affects everything."
The survey found that 66% of respondents believed that investment in the South West's road infrastructure is poor or very poor and 65% felt the same about the region's rail investment. Transport links to London and major airports for international travel were of particular concern.
