Labour deputy leader Angela Rayner, in her first Prime Minister's Questions, urged Mr Johnson to "get some skates on" before a "winter crisis hits" the UK's care homes.
She said the UK is "staring down the barrel of a second wave" and care home workers are "still not getting the PPE they need, they're still not getting the testing they need."
The care sector has been severely hit by the coronavirus crisis, with over 20,000 residents having died with the illness.
Ms Rayner added: "I urge the Prime Minister to get on top of this problem now before the winter crisis hits.
"The Prime Minister has put his faith in Operation Moonshot, but meanwhile on planet Earth there are no NHS tests available for several high-infection areas."
The prime minister acknowledged concerns about Covid-19 infection rates in care homes and said he was preparing to announce a "toughening up" of the regulations governing them.
"We are concerned about the rates of infection in care homes, clearly they've come down massively since we instituted the £600 million care home action plan.
"Tomorrow we will be announcing a further winter care home action plan and it won't surprise her to know that we want to see a toughening up of the rules governing the movement of workers from one care home to another, we want to make sure we protect care homes from further infections."
Ms Rayner told the PM she'd been forced to stand in for leader Sir Keir Starmer as he had been self isolating while waiting for a coronavirus test result from a family member.
She said: "Keir wasn't able to go to work today and his children couldn't go to school because his family had to wait for their coronavirus test results despite the Prime Minister's promise of results within 24 hours. "
The Labour leader's child, who was experiencing coronavirus symptoms, was given the all clear 20 minutes before PMQs but still let Ms Rayner take his place.
Mr Johnson defended the speed of testing, and asked why Sir Keir was absent from PMQs, given he'd left isolation.
Sir Keir, whose child was able to get a swift coronavirus test because his wife works in the NHS, also took aim at the government over the testing system.
He wrote on Twitter: "The situation is desperate at the moment for thousands of families across the country who are struggling to get a test.
"They deserve answers and for this problem to be fixed."
The government has been heavily criticised over the testing system, with some people having to wait days for a result and others being told to drive for hours to get a test.
In a dig at the PM's chief adviser Dominic Cummings, Ms Rayner said: "Next time a man with Covid-19 symptoms drives from London to Durham it will probably be for the nearest Covid test."
The prime minister said issues with testing were down to a "huge surge in demand" and he urged people to only book a test if they have symptoms.
It was the first time Ms Rayner faced the prime minister across the Despatch Box.