Born in Rotterdam, she started swimming under the coaching of "Ma" Braun, who had coached her daughter to an Olympic gold medal in 1928. In 1934, Mastenbroek won three gold medals and a silver at the European Championships.
She repeated that performance at the 1936 Summer Olympics, aged only 17, winning the 100 m freestyle, 400 m freestyle and the 4 x 100 m freestyle. In the 100 m backstroke, she finished second behind teammate Nida Senff. (Senff missed a turning point and had to swim back before completing the last 50 m. She nevertheless beat Mastenbroek thanks to an outstanding last leg.)
The following year she became a swimming instructor, thereby losing her amateur status and becoming ineligible for competition.
During her career she broke nine world records (six for backstroke; three for freestyle). In 1968 she was inducted into the International Swimming Hall of Fame.
She passed away at age 84 in Rotterdam.