A drug testing team from UK Sport was asked by the WRU to carry out a series of tests on the Penygraig players following information passed on to the Union by the police.
A spokesperson for UK Sport confirmed there was "a blanket refusal" by the players from Penygraig to take the tests and that they left the ground without taking any samples.
"We were asked by the WRU Group Chief Executive David Moffett, via our colleagues at the Sports Council for Wales, to carry out tests on the Penygraig players at the end of Wednesday night's game," said the UK Sport spokesperson.
"The collective actions of Penygraig RFC constitute a prima facie breach of rugby's regulations. We informed them of this fact and told them that a refusal to submit to a test is deemed to be an offence.
"Despite that, we were unable to complete the task we had been asked to undertake by the WRU and are in the process of submitting a report to them to that effect."
As a result of the information the WRU has received from UK Sport, Mr Moffett has informed the chairman of the WRU National League Management Committee of the facts and the club and its players will now be subject to the normal disciplinary procedures in these cases.
"This is a very serious situation. The WRU received information from the police alleging that some players might be using Class A drugs and, as a result, I considered it appropriate to ask UK Sport to invite the whole of the Penygraig team to submit to formal testing procedures," said Mr Moffett.
"I attended the fixture along with other representatives of the WRU and witnessed at first-hand the events of the night. The WRU takes very seriously its obligations to rugby union, and sport as a whole, in ensuring that the use of illegal drugs plays no part in the game."
This matter is now being reviewed collectively by the WRU, Sports Council for Wales and the South Wales Police, who have forged a working partnership to help fight against the threat of drug abuse in sport.
South Wales Police Assistant Chief Constable Dave Francis said: "Following receipt of information about allegations of the use of performance-enhancing drugs at Penygraig Rugby Club, South Wales Police shared its concerns with the WRU, who took the decision to carry out drug-testing at Wednesday night's game, in line with its on-going efforts to tackle drugs in the sport,".
"We are aware that the Penygraig rugby club chose not to undertake those tests and it is a matter for the WRU to determine what action, if any, it will now take."