Cucurbitacins are seldom found in pickling cucumber, but when present, make finished products unpalatable. Liquid chromatography-time-of-flight mass spectrometry was employed to determine cucurbitacins in ‘Hanzil’ (bitter) and ‘Vlaspik’ (non-bitter) pickling cucumbers subjected to acidification or fermentation. Cucurbitacins C (CuC) and C-glycoside were putatively identified in ‘Hanzil’ cucumber fruits while undetectable in ‘Vlaspik’. The major cucurbitacin in ‘Hanzil’, CuC, increased in concentration with cucumber size, ranging from 28 ppm in size 2 A to >74 ppm in size 4+ fruits. Endocarp had the highest CuC concentration (88.7 ± 44.3 ppm) followed by mesocarp (30.6 ± 15.1 ppm) and exocarp (2.58 ± 1.75 ppm). CuC was 3-fold lower in acidified cucumber (15.7 ± 10.3 ppm) and 10-fold less in fermented cucumber (4.90 ± 3.94 ppm) than when fresh (47.9 ± 22.8 ppm). Fermentation has potential for de-bittering of cucurbits, and oversized bitter cucumbers could serve as a source of CuC for exploration of bioactivities.