Bee drone brood is a beehive by-product with high hormonal activity used in natural medicine to treat male infertility. The aim of the study was to assess the effect of drone brood on stallion spermatozoa during a short-term incubation for its potential use in the equine semen extenders. Three different forms of fixed drone brood (frozen (FR), freeze-dried (FD), and dried extract (DE)) were used. Solutions of drone brood were compared in terms of testosterone, protein, total phenolic content, and antioxidant activity. The stallion semen was diluted with prepared drone brood solutions. The computer-assisted semen analysis (CASA) method was employed to evaluate the movement characteristics of the diluted ejaculate. To determine spermatozoa viability, the mitochondrial toxicity test (MTT) and Alamar Blue test were performed. In terms of testosterone content and antioxidant activity, a close likeness between FR and FD was found whereas DE’s composition differed notably. FR had a positive effect mainly on progressive motility, but also on sperm distance and speed parameters after 2 and 3 h of incubation. On the contrary, FD and DE acted negatively, depending on increasing dose and time. For the first time, a positive dose-dependent effect of fixed drone brood on spermatozoa survival in vitro was demonstrated.