If the league determines the Chargers used the substance, they could be fined or lose a draft pick.
Fox Sports first reported the investigation on Sunday.
"We have been looking into it," NFL spokesman Greg Aiello said.
The Chargers issued a statement Sunday that sad they are "aware of the inquiry and are cooperating fully with the League."
During Monday night's game, an equipment manager came onto the field with an illegal substance on hand towels, Fox Sports reported. Line judge Jeff Bergman saw the towels and tried to confiscate the substance. When the equipment manager wouldn't give it up, the officials made him empty his pockets and found a skin colored or clear type of tape, Fox Sports reported.
The Chargers blew a 24-0 halftime lead and lost 35-24, their second straight loss in which they blew a double-digit second-half lead.
It wasn't clear whether the Chargers were on offense or defense when the substance was confiscated.
In 1981, the NFL banned the product Stickum and other sticky substances. Raiders cornerback Lester Hayes was renowned for using the substance before it was banned.
News of the investigation capped one of the most tumultuous weeks in club history. Three days after the embarrassing loss dropped the team to 3-3, club spokesman Bill Johnston infuriated many fans when he posted a column on the team's web site that began by saying, "What's up with you people?" and told fans that it was "Time to take a chill pill."
Fans have been unhappy with coach Norv Turner and general manager A.J. Smith, who were brought back in January despite the Chargers missing the playoffs for the second straight season. San Diego has just one playoff win in four seasons.