Emergency services descended on Lichfield Street outside The Moon Under Water Wetherspoon pub last night after a "strong chemical smell" was reported.
It is believed a customer set off a CS gas canister in the pub opposite Wolverhampton Grand Theatre at around 8.40pm
Six people were treated after reporting breathing difficulties but nobody needed hospital treatment and all were discharged at the scene.
CS Gas is illegal in the UK but so far no arrests have been made.
Around 800 theatregoers emerging from The Grand, which was hosting the opening night of musical Fame, were quickly ushered away from the scene as officers prepared to search the pub just after 10pm.
West Midlands Police officers were joined by fire and ambulance crews, including two major incident units, and the road was cordoned off from Pipers Row to Princess Street as an investigation was launched.
A West Midlands Police spokeswoman said: "Police are investigating after a substance believed to be CS spray or similar was let off in a pub in Wolverhampton.
"A cordon was put in place while the area was made safe."
The Moon Under Water pub reopened this morning and Wetherspoon said staff were helping with the police investigation.
A JD Wetherspoon spokesman said: "We can confirm that a customer let off a CS gas canister in The Moon Under Water last night."
They added: "The police are investigating and have clear CCTV footage of the incident.
"The pub remained closed last night, but has reopened as usual this morning."
Crews from Wolverhampton and Fallings Park fire stations attended and set up the initial cordon, said a West Midlands Fire Service who added: "The pub had been evacuated by staff prior to fire service arrival.
"A cordon was established initially by the fire service, assisted by police and the premises was investigated by fire service personnel wearing breathing apparatus using gas monitors.
"After investigations, in which no smells or abnormal readings were detected, our crews ventilated the premises.
"Six members of the public who had complained of breathing difficulties and nausea were checked by ambulance, their conditions improved with fresh air and they were released from the scene.
"A controlled release of around 800 customers from the Grand Theatre opposite was conducted by police to safely move customers out of the cordon area."
A spokesman for the Grand Theatre said: "That evening’s performance of Fame was unaffected and the theatre management would like to commend the professionalism of their staff and emergency services.”
A West Midlands Ambulance Service spokesman added: “On arrival we discovered six patients who had been exposed to an unknown substance.
"Following assessment from ambulance staff, none of the patients required hospital treatment and they were all discharged at the scene.”
Police enquiries are ongoing and anyone with information can call 101 quoting log 2440 of November 19.