This study aimed to examine how varying concentrations (0.5%, 1.0%, 2.0%, 3.0%, and 4.0%) of nano-L-α phosphatidylcholine (nano-L-α-PC) affected the quality of post-thawed buffalo bull semen compared to the same concentrations of L-α-phosphatidylcholine (L-α-PC), while 20% egg yolk (EY) was used as a control. Ejaculates (n = 72) were collected from fertile buffalo bulls (n = 12) for 6 weeks. The ejaculates were pooled and divided into 11 groups (3 aliquots per group) extended with 20% EY (Group 1 without supplement), 0.5%, 1.0%, 2.0%, 3.0%, and 4.0% nano-L-α-PC (Groups 2–6), and 0.5%, 1.0%, 2.0%, 3.0%, and 4.0% L-α-PC (Groups 7–11) in tris buffer. After cryopreservation procedures, semen samples were thawed at 37 °C and then evaluated for sperm kinematics, acrosomes, plasma membranes, and DNA integrities. The seminal plasma was analyzed for malondialdehyde (MDA), superoxide dismutase (SOD), glutathione reduced (GSH), and catalase (CAT). Results demonstrated that extended semen samples containing 1.0-3.0% nano-L-α-PC and 1.0-4.0% L-α-PC showed high total motility compared to control, whereas at 0.5–4.0% of nano-L-α-PC and L-α-PC showed high progressive motility. Significant (P< 0.05) wobble levels were seen at 0.5, 1.0, and 3.0% nano-L-α-PC, and distance average path and velocity average path at 2.0% L-α-PC. Acrosome and plasma membrane integrities were markedly elevated (P< 0.0001) at 2.0% nano-L-α-PC and 4.0% L-α-PC. High DNA integrity metrics were noticed at 0.5–1.0% nano-L-α-PC, as well as 3.0% and 4% L-α-PC. Low MDA and high (SOD, GSH, and CAT) levels were observed at 2.0% nano-L-α-PC and 4.0% L-α-PC. In conclusion, the optimum concentration of nano-L-α-PC that improved semen quality was 2.0%, which is roughly equivalent to the effect of 4.0% L-α-PC.