Patrolling officers spotted a white car arrive outside Bar Bambi in Flinders Lane in Melbourne's CBD about 4.45am on Friday.
The pair, both 16, allegedly ran from the car holding a red jerry can before smashing a window of the licensed venue and attempting to pour fuel inside.
Observing officers intervened and arrested the two boys as the driver fled in the car, which has not been located.
The teens have both been charged with attempted criminal damage by fire, reckless conduct endangering life, burglary and handling stolen goods.
Detectives are working to identify possible links to previous incidents.
A single red jerry can sat outside the empty venue in ACDC Lane, which remained taped off by police.
State Opposition Leader Jess Wilson held a private forum at the venue where she described the hospitality sector as the "heart and soul of city" less than 24 hours before the latest attack.
The popular nightlife venue has been targeted by alleged arsonists twice before, including on April 25 when three teens were charged over an alleged attack.
"Venue owners want more police and tougher laws. That's why, under a government I lead, we will recruit 3000 more police, implement real adult time for adult crime, and introduce one-strike bail laws" she said on Friday.
Separately, police arrested two teens over the alleged firebombing of The George Hotel in South Melbourne after five Molotov cocktails were thrown towards the venue on May 2.
No one was inside at the time and the fire was put out quickly by responding authorities, leaving minimal damage.
Detectives raided two homes on Friday morning when they seized a gun, several electronic devices and a quantity of drugs.
They arrested two 17-year-old boys.
Officers have also arrested and charged a Tarneit man over an investigation into extortion demands on two other venues in Melbourne's CBD.
The 20-year-old allegedly made two demands for $10,000 to venues on Flinders Lane and Lonsdale Street over social media on May 7.
Detectives seized several knives, ammunition and a phone from his home before charging him with weapons-related offences.
He was bailed to appear at Wyndham Magistrates Court on June 12.
Investigations are ongoing into whether the alleged extortions are linked to the hospitality venue attacks or whether they involved opportunistic people trying to take advantage of the hospitality industry while they're down, Detective Superintendent Jason Kelly said.
Victoria Police have increased patrols around Melbourne's entertainment precincts following the spate of attacks on licensed premises.
Operation Eclipse was set up to investigate the incidents believed to involve organised crime syndicates, as investigators search for the organisers and those pulling the strings.
To date, officers have arrested 51 people over attacks on the Melbourne hospitality industry, with 38 of those achieved "on the hop" as people were about to commit the offences.
