It was the first time Spain's powerful banking sector, recovering after a crisis that brought it close to collapse, has spoken out in the delicate political stand-off between Catalonia and Madrid.
Catalan leaders are campaigning for independence in a regional election on September 27, a move opposed by the central government in Madrid and some business groups.
Major European allies have warned the region it would drop out of the EU if it seceded from Spain. The European Commission reiterated that stance on Thursday, raising pressure on the leader of the independence drive, Catalonia's regional president Artur Mas.
On Friday the banking associations AEB and CECA joined the fray, warning in a statement of "risks to financial stability" that would force them to "reconsider" their presence in Catalonia, which would cause a shortage of credit.
The Spanish Banking Association (AEB) groups dozens of big Spanish lenders including BBVA and foreign ones active in Spain including Barclays, Citibank and HSBC.
CECA, the Spanish Savings Banks Confederation, groups a handful of Spanish entities such as Bankia and Catalan lender Caixabank.
Some of the banks only have branches in Catalonia but major ones such Caixabank and Sabadell are based there.
Polls show pro-secession candidates could win a majority of seats in the Catalan parliament in next weekend's election.
If they win, Mas has vowed to push through an 18-month roadmap to secession for the region, which accounts for a fifth of Spain's economy.
Catalonia is the biggest political challenge to Spain's conservative Prime Minister Mariano Rajoy as he prepares to fight for re-election in December.
He has called for Spain to be unified as it recovers from an economic crisis and says secession would be illegal.
Ahead of the election, AEB and CECA highlighted "the risks that would be posed to financial stability by any political decision what would break current laws and lead to part of Spain being excluded from the European Union and the eurozone," they said.
"The exclusion of Catalonia from the eurozone as a result of unilaterally breaking away from the current constitutional framework, would pose serious legal problems to all banks with a presence in Catalonia," it added.
"These difficulties would oblige those entities to reconsider the strategy of their presence there."
The business and finance world had kept largely silent in public over the mounting tension between Barcelona and Madrid, but key figures have spoken out recently.
The president of Catalonia's main business association Foment del Trabell has warned that secession could cause "enormous financing difficulties".
"For us this creates tension and great worry because we do not want these processes to disrupt the economic recovery," he told AFP in an interview.
He has called on the Spanish government to negotiate over Catalan demands and to grant the region greater fiscal autonomy and cultural recognition.
Spain's Finance Minister Luis de Guindos moved to calm jitters over Catalonia in an interview published in the Financial Times on Friday.
"My message to investors is that independence will not take place," he said.
He argued that the real risk to business in Catalonia was not independence but the rise of left-wing elements running in an electoral alliance with Mas.
The Madrid stock exchange fell sharply by around three percent after the announcement. It closed 2.57 percent lower at 9,847 points on Friday.