Here's what happened ... the tennis legend walked onstage with her sister, Venus, and actress Quinta Brunson to honor women's sports during the event at the Dolby Theater in Los Angeles.

While they were addressing the crowd, Venus said, "So, go ahead and enjoy women's sports like you would any other sports, because they are sports."

As Venus was talking, Serena was giggling before turning more serious and suddenly blasting the Kansas City Chiefs kicker ... "Except you, Harrison Butker. We don't need you."

Brunson chimed in ... "At all. Like, ever."

Earlier, Butker was photographed arriving in a suit and bow tie to the awards show. It's unclear what Butker's reaction was to Serena taking a swipe at him.

You may recall ... Butker made headlines in May when he gave the commencement speech at Benedictine College in Kansas, encouraging female graduates to become homemakers who raise children. The national backlash was immediate, especially from professional women in the workplace.

During his speech, Butker -- a staunch Catholic -- also denounced the LGBTQ community and criticized President Joe Biden's stance in favor of abortion.

Despite all the negativity, Butker defended his views and was unapologetic about his love for Catholicism and traditional American values.

Meanwhile, Serena -- who hosted the ESPYs -- gave a funny monologue about WNBA phenom Caitlin Clark at another point in the show.

She joked, "You are the Larry Bird in that you are an amazing player, you have ties to Indiana, and white people are really crazy about you."