Fish sperm cryopreservation is important for preserving sperm viability and genetic potential during freezing and thawing. Each fish species requires a specific extender because of diverse sperm characteristics. Tilapia (Oreochromis sp.) popular in Malaysia for its hardiness and rapid growth is an excellent choice for aquaculture. Aloe vera (Aloe barbadensis) (Av) shows promise in spermatogenesis, but its use as a cryoprotectant in semen extenders is still underexplored. This study investigates the effects of antibiotic- and sugar-free Av-based extenders on cryopreserved tilapia sperm. Semen samples were collected and divided into four treatment groups: Control (C, 0% Av), Treatment 1 (T1, 10% Av, antibiotic: sugar), Treatment 2 (T2, 10% Av, antibiotic: sugar-free), and Treatment 3 (T3, 10% Av, antibiotic-free: sugar) mixed with a Tris stabilizer. The samples were loaded into French straws and were subjected to an equilibration phase before being frozen and stored in liquid nitrogen. The straws were thawed after 48 hours to evaluate the effect of extender components. After cryopreservation, T1 showed significantly highest viability with 63.4±3.0%. The improved viability in T2 likely resulted from the antibiotics while the reduced viability in T3 may be due to their absence, with values of 45.7±1.9% and 36.6±1.8%, respectively. After 48 hours of storage, T1 exhibited decreased motility with most spermatozoa displaying strong vibration. In contrast, T2 and T3 experienced a greater decline in motility with a weaker vibration. Antibiotics in T2 likely contributed to the higher motility observed in this treatment. In summary, the absence of antibiotics and sugar negatively affected sperm viability and motility. The addition of antibiotic, sugar, and Av in the extender proved crucial for maintaining the viability and motility of cryopreserved tilapia sperm. These research findings enhance a better understanding of the potential of Av-based extenders in fish sperm cryopreservation that contribute to sustainable food security.