The 23-year-old clocked 23.55 seconds, breaking the previous record of 23.59 set by fellow American Kate Douglass earlier this month at a US Pro Swim Series event in Indianapolis.
In May, Walsh bettered her own 100 metres butterfly world record for the third time in a year, posting 54.33 seconds in Fort Lauderdale, Florida.
With the 50m freestyle mark, Walsh has now broken a world record 21 times.
She also got two relay gold medals at the 2024 Paris Olympics and won her first individual long-course world title in July last year at the World Aquatics Championships in Singapore, where she took gold in the women's 100 metres butterfly.
"I would feel a lot of regret if I left the time like this on the table and never was able to go (at) it again, so I say the more the merrier," Walsh told swimming news outlet SwimSwam after breaking her newest record.
"I'm going to keep trying to break world records in everything because that's what it takes to be at the level that I'm at."
Walsh is viewed as a top contender for the US heading into the 2028 Los Angeles Olympics, with multiple world records and a world championship already to her name.
"Maybe some eyes will be on me more so than ever now, and I'm ready for the challenge to handle that pressure, especially at a home Olympics, so I'm here, bring it on!" she added.