A total of 996 players and staff were tested for COVID-19 across Tuesday, Thursday and Friday, with the positive results coming from different clubs.
They will now be required to self-isolate for a seven-day period.
The findings are in addition to the six who were found to have contracted the virus in the first round of testing, which covered 748 people, on 17 to 19 May.
A Premier League statement read: "The Premier League can today confirm that on Tuesday 19 May, Thursday 21 May and Friday 22 May, 996 players and club staff were tested for COVID-19. Of these, two have tested positive from two clubs.
"Players or club staff who have tested positive will now self-isolate for a period of seven days.
"For the second round of testing, the number of tests available to each club was increased from 40 to 50.
"Previously, 748 players and club staff were tested for COVID-19 on 17 to 19 May, with six testing positive from three clubs.
"The six who tested positive in the first round of testing are not included in the figures for 19 to 22 May, as they are still within their seven-day self-isolation period.
"The Premier League is providing this aggregated information for the purposes of competition integrity and transparency. No specific details as to clubs or individuals will be provided by the League and results will be made public after each round of testing."
The Premier League is yet to announce any date for a restart to the competition, which has been stalled since March as a result of the pandemic.
Organisers have said there are still hopes of a resumption in June, but have also admitted abandonment of the 2019-2020 season remains a realistic possibility.
The Bundesliga was the first of Europe's big five leagues to restart its season after France's Ligue 1 decided to finish its season early. LaLiga announced on Sunday (AEST) that it has been cleared to start again from 8 June, and Serie A is expected to follow barring any exceptional circumstances.