Regional police said four people were arrested when officers met resistance while trying to clear groups of protesters who had stopped traffic. The regional emergency service said 37 people were treated for minor injuries.
Twelve officers were also injured in the clashes, according to police. Protesters threw rocks at police lines and burned tires on some highways.
Regional transportation authorities said the protests disrupted traffic on main thoroughfares in Barcelona and affected travel on a half-dozen major highways and train lines elsewhere in Catalonia.
The general strike was organized by small unions of pro-independence workers and students. On paper, they were demanding improved social policies, including a 35-hour work week and a higher minimum wage.
However, the protesters carried pro-secession flags and chanted slogans for the release of the 12 separatists who are on trial in Spain's Madrid-based Supreme Court for their roles in a 2017 independence declaration.
The major labor unions in Catalonia did not back Thursday's strike, which appeared to have a limited impact on businesses.
In Barcelona, the region's capital, students in favor of secession held a mid-day march attended by 13,000 people, according to city police.
Tens of thousands more joined an evening march behind a banner that read "Self-determination is not a crime." A grassroots pro-secession group, ANC, organized the march.
The Spanish government says regions cannot independently secede, according to the Constitution.
The 12 Catalan separatists are charged with sedition and other alleged crimes for their roles in Catalonia's failed 2017 secession attempt. Three were scheduled to testify in court on Thursday.
Their trial is in its second week and is expected to last three months.
Election results and polls indicate that Catalonia's 7.5 million residents are divided down the middle over the secession issue.
AP journalist Aritz Parra contributed to this report from Madrid.
More AP coverage of Catalonia at https://apnews.com/Catalonia