Putellas, 29, scored in the 51st minute from a Patri Guijarro cross, bringing her total for the club to 182 in 404 games.
Hermoso, who now plays for Pachuca in Mexico, formerly held the record with 181 goals in two stints with Barça, between 2013 and 2017 and 2019 and 2022.
In Madrid, Barça dominated the first half but failed to push home keeper Lola Gallardo into a noteworthy save.
Guijarro came close to scoring early in the second quarter when Gallardo tipped her effort around the post only minutes before setting up Putellas to open the scoring.
Substitutes Salma Paralluelo and Asisat Oshoala had chances to score a second, but keeper Cata Coll made late saves on Sheila Garca and Leicy Santos as Barça won their fifth game in Liga F this season.
They are joint top of the standings with Real Madrid, who also have a maximum of 15 points, but Atletico Madrid has dropped five points behind the leaders after suffering their first defeat of the season.
Putellas’ winning goal was her third in three games as she recovers from an ACL injury.
She returned to the World Cup with Spain after missing practically the full season, despite being out of match shape as her country won the event in Australia and New Zealand.
Putellas returned to Barça in 2012 after spending a year as a kid at the Catalan club.
She has now made over 400 games for the club, earning seven league crowns and two Champions Leagues, among other team and individual honours.
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