In this study, the fractional factorial design of experiments (DoE) method was applied to analyze the influence of process variables on the thermoforming of film used for disposable medical device packaging in a real industrial environment. The study investigated how process parameters, tool geometry, and film thickness affect variations in the thickness of the thermoformed material. The conducted experiments demonstrated that the impact of individual parameters depends on the specific combination of film and tooling used, and that interactions between process variables exhibit a nonlinear nature. The adjusted coefficient of determination R2(adj) did not exceed 40% for thicker film, regardless of the tooling used, suggesting that a significant portion of the variance in results was not explained by the analyzed factors. Moreover, ANOVA analysis indicated that the significance of process parameters varied depending on the experimental setup. The application of the DoE method in an industrial setting is limited due to economic constraints, as it requires a substantial number of experiments and measurements to fully determine the influence of process variables on the quality of thermoformed film. The results highlight the need for alternative analytical methods capable of capturing the nonlinear relationships present in the thermoforming process. In the future, machine learning methods are planned to be employed for more effective process modeling.