In plant matrices, folates exist largely as folylpoly-γ-glutamates requiring deglutamylation to monoglutamates prior to absorption, which might impair dietary folate bioavailability. This study investigated folylpoly-γ-glutamate stability and conversions in broccoli tissue during thermal (25–90 °C, 30 min) and high-pressure treatments (0.1–600 MPa, 25–45 °C, 30 min) after vacuum packaging. Folates were analyzed based on poly-γ-glutamate side chain length by RP-HPLC. During thermal treatments, folates were stable up to 90 °C, whereas differences in folylpoly-γ-glutamate profiles towards higher conjugated folylpoly-γ-glutamates were observed at elevated temperatures (70–90 °C). High-pressure treatments resulted in significant folate losses (48–78%). Depending on the pressure–temperature combinations studied, folylpoly-γ-glutamates were converted to folylmono- and folyldi-γ-glutamates, which was shown to occur mainly during the initial stages of the high-pressure treatments, i.e. during pressure build-up and subsequent equilibration. Targeted application of high-pressure treatments can hence be applied to obtain broccoli with higher monoglutamate folate content. Implications towards folate bioavailability in relation to the observed folate degradation, however, requires further investigation.