Seasonal fluctuations of climate are considered a major factor affecting spermatogenesis and semen quality in the bovine. Our study aimed to investigate the effect of season on functional parameters of frozen-thawed bovine semen using computer-assisted sperm analysis (CASA) and flow cytometry. For this purpose, 86 ejaculates were collected from five mature Holstein-Friesian bulls kept under subtropical conditions during summer (August to September; n = 43) and winter (December to January; n = 43) months. Semen was diluted with a Tris-egg yolk-based extender and frozen at −196 °C. Computer-assisted sperm analysis was performed immediately after thawing (0h) and after 3 hours of incubation (3h) to evaluate the percentage (%) of total motile, progressively motile, and rapidly motile sperm. In addition, the average path, curvilinear, and straight-line velocities as well as the amplitude of lateral head displacement of sperm were determined. The percentages of sperm with intact plasma membrane and acrosome (PMAI, %), with high mitochondrial membrane potential (HMMP, %), with low intracellular Ca+2 levels (LOW-Ca+2, %), and with high DNA fragmentation index (DFI%, %) were flow cytometrically determined at 0 and 3h. The survival rate of sperm under hypotonic conditions (HYPO-SURV, %) and the percentage of sperm with inducible acrosome reaction (IAR, %) were assessed using flow cytometry at 0 and 3h, respectively. The fixed effect of season (winter vs. summer) on the quality parameters of sperm was explored by applying linear mixed-effects models. The results showed an improvement of all CASA parameters, except for the straight-line velocity (P > 0.05) in winter compared with summer for both unincubated and incubated sperm (P < 0.01 in all cases). Ejaculates collected in summer had lower values of IAR (P < 0.001) as well as PMAI, HMMP, and LOW-Ca+2 at 0 and 3h (P < 0.01 in all cases). On the contrary, HYPO-SURV and DFI% (at 0 and 3h) were not affected by season (P > 0.05 in all cases). Concluding, the employment of CASA and flow cytometry revealed season-related alterations in the functional status of cryopreserved bovine sperm, which suggest an adverse effect of summer heat stress on motility, plasma membrane and acrosome integrity, inducibility of acrosome reaction, mitochondrial function and intracellular Ca+2 content, but not on the DNA integrity of sperm after freezing–thawing.