Broadly neutralizing antibodies are produced in 15–25% of the infected individuals after a minimum of 2–3 years of HIV infection (34). Previously, we have for the first time showed the presence of cnAbs targeting multiple HIV-1 epitopes in the plasma of HIV-1-infected children (35, 36). Later, the development of bnAbs in infants was reported by Goo et al. (37) followed by successful isolation of a bnAb BF520.1 directed against N332 V3-glycan epitope from an infant (31). In a recent study, we observed the evolution of cnAbs with multiple epitope specificities in the plasma of chronically infected pediatric donors (38) who were anti-retroviral naïve for ≥5 years of infection and potential long-term non-progressors, identifying them as suitable candidates for the isolation of bnAbs. The CD4bs on the viral envelope glycoprotein gp120 is a highly conformational epitope. Single chain antibody fragments (scFvs) retain their antigen binding sites and yet are small and flexible to overcome such constraints posed by conformational embedded epitopes. Phage display has proved to be a powerful method for the generation of recombinant antibody fragments (39, 40), its main advantage being the large diversity of antibodies with varied epitope specificities that can be generated by biopanning with different antigens (41–44). Therefore, this study was aimed to construct a peripheral blood mononuclear cell (PBMC) based scFv phage library, from nine subtype C infected antiretroviral naïve pediatric cross-neutralizers, whose plasma antibodies exhibited potent and cross-neutralizing activity, to identify scFvs against the CD4bs region of subtype C HIV-1. The phage library comprised of 9 × 108 clones and further biopanning this library using CD4bs specific antigen RSC3 core protein, led to the identification of two cross-neutralizing anti-CD4bs scFv monoclonals. The pediatric anti-CD4bs scFv monoclonal 2B10 exhibited cross neutralization activity against subtype C viruses of Indian and African regions and with other non-subtype C viruses.