Mannose-binding lectin (MBL) is an important glycoprotein of the human innate immune system. Furthermore, individuals with sickle cell anemia (SCA) and MBL deficiency seem more susceptible to vaso-occlusive crises, suggesting an MBL role on HbSS red blood cells (RBCs). This study investigated the interaction of MBL with HbA (healthy) and HbSS RBCs using optical tweezers (OT) and atomic force microscopy (AFM). OT was employed to measure the RBC overall elasticity, while AFM was applied to assess the local stiffness and roughness of the cell membrane. Osmotic fragility assays were also performed. Additionally, cationic quantum dots (QDs) were applied to evaluate the membrane charges of HbSS RBCs. Results using QDs indicated that HbSS RBCs have a less negatively charged surface than HbA RBCs, potentially due to a reduced sialic acid content. Osmotic fragility assays showed that MBL enhanced the resistance of RBCs to lysis, while OT and AFM indicated a decrease in their elastic capacity following MBL incubation. Moreover, OT and membrane roughness results suggested a greater interaction of MBL with HbSS RBCs. We believe this study provided insights into the MBL interaction with HbSS RBCs, inciting further investigations to better understand its involvement in SCA pathophysiology in vivo.