Footage published by The Sun allegedly shows the 29-year-old Mali international using the substance. A club spokesperson confirmed that Spurs are looking into the situation and will handle it internally.
Bissouma previously faced a one-match ban in 2023 after being pictured with the same substance and later apologised, admitting he showed poor judgement.
In the UK, using nitrous oxide for fun has been illegal since 2023 and can carry a prison sentence of up to two years.
Bissouma, who joined Tottenham from Brighton in a £30 million deal in 2022, is in the final year of his contract. He has not featured since Thomas Frank was named manager in June.
He was also left out of the squad for the Uefa Super Cup match against Paris Saint-Germain in August due to repeated lateness and has struggled with ankle and knee injuries this season.
