The aim of this study was to examine the effectiveness of cryomilling (CM) pretreatment on the isolation of fennel seeds essential oil (EO). Therefore, the study included (i) the process optimization and comparison of the efficiencies of hydrodistillation (HD) and steam distillation (SD); (ii) the evaluation of the effect of the CM application prior to the distillation method (selected based on the results obtained in the first part) on the EO yield; (iii) the evaluation of possible quality differences of the EOs obtained with the performed isolation methods. The obtained results showed that HD (at a solid to liquid ratio 1:10 and a distillation time of 120 min) was more efficient in terms of the EO yield compared to SD (at a pressure of 0.83 bar and distillation time of 117 min as optimal conditions). Moreover, an increased EO yield or even reduced distillation time was observed when HD was combined with a 3 min or 5 min CM pretreatment. GC-MS analysis showed no qualitative differences in chemical composition upon any of the applied isolation procedures, although higher amounts of volatiles were found in the cryomilled samples. The results of this study could be of interest to academia and the EO industry, as CM showed a positive aspect in EO isolation that could provide economic benefits in terms of higher yields or energy savings.