Objective Short gamete co-incubation is used in conventional in vitro fertilization (IVF) protocols to reduce the duration of oocyte-sperm exposure and enable earlier identification of fertilization failure. However, laboratory conditions associated with fertilization outcomes under short co-incubation remain insufficiently characterized. This retrospective study evaluated whether different combinations of sperm preparation and fertilization media were associated with fertilization outcomes during short gamete co-incubation in conventional IVF. Methods This single-center retrospective study screened 216 insurance-covered conventional IVF cycles comprising 1,591 oocytes performed between August 2023 and July 2025. After application of exclusion criteria, 738 mature oocytes from 108 cycles were included in the analysis. Mature oocytes were defined as those exhibiting at least one polar body after IVF. Cycles were categorized into three medium-combination groups according to the laboratory protocol period in which each combination was used: Group A (Iso ×ばつ Gx-IVF; January 2024 to March 2025), Group B (Gra ×ばつ Gx-IVF; August to November 2023), and Group C (Gra ×ばつ SF; April to July 2025), with December 2023 treated as an excluded mixed-medium transition period. Outcomes were analyzed per mature oocyte. The primary outcome was two-pronuclear (2PN) fertilization. Secondary outcomes included 0PN, 1PN, ≥3PN, and the proportion of good-quality embryos on Day 2. Multivariable logistic regression models with cycle-clustered robust standard errors were used to account for intra-cycle correlation among oocytes. Results The 2PN fertilization rate differed significantly among the three groups: 43.0% in Group A, 59.8% in Group B, and 66.9% in Group C (p < 0.001). The 0PN rate was highest in Group A and differed significantly among groups: 49.8% in Group A, 30.8% in Group B, and 21.5% in Group C (p < 0.001). The proportion of good-quality embryos on Day 2 was 33.2%, 41.9%, and 42.1% in Groups A, B, and C, respectively; however, this difference did not reach statistical significance (p = 0.067). In cycle-clustered multivariable logistic regression models, Group B was associated with significantly higher odds of 2PN fertilization than Group A after adjustment for patient characteristics, semen parameters, and time-related laboratory factors (adjusted odds ratio (aOR), 2.307; 95% confidence interval (CI), 1.165-4.572; p = 0.017). Group C showed a similar trend toward higher odds of 2PN fertilization, although the association was borderline after full adjustment (aOR, 2.738; 95% CI, 1.001-7.484; p = 0.050). Conclusion In this single-center retrospective study, sperm preparation and fertilization medium-combination protocols were associated with 2PN and 0PN fertilization outcomes during short gamete co-incubation in conventional IVF. Compared with Group A, Group B demonstrated higher adjusted odds of 2PN fertilization after controlling for patient characteristics, semen parameters, and time-related laboratory factors, whereas Group C showed a similar trend with borderline statistical significance after full adjustment. These findings are limited to fertilization outcomes and should be interpreted as hypothesis-generating because of the retrospective design, non-randomized implementation across different calendar periods, and lack of downstream reproductive outcomes. Prospective studies are warranted to confirm these observations.