This study investigates the an eco-friendly synthesis for the production of titanium dioxide nanoparticles (TiO2 NPs) by employing the water-soluble flower extract of Calendula officinalis as a natural reducing and stabilizing agent. The green produced TiO2 NPs/C. officinalis were characterized using different advanced analytical techniques, including FT-IR, FE-SEM, EDS-elemental mapping, XRD, ICP-OES and TEM. It represents the TiO2 NPs were found to be spherical in shape and well distributed without agglomeration. It also revealed the particle sizes were in the range of 30–50 nm. EDX represented the Ti atomic species along with C and O are seen to be well-dispersed in a homogeneous manner. The broad absorption band appeared at 3200–3450 cm−1 assigned to OH bond stretching belong to the adsorbed water over the titanium dioxide nanoparticles. Further, newly synthesized TiO2 NPs/C. officinalis are used as a nanocatalyst for the reduction of 4-nitrophenol (4-NP). A UV–Vis spectrophotometer is employed to track the effectiveness of the reduction process. Recyclability in 8 further degradation cycles is used to assess the sustainability of this catalyst, and the results show a steady and notable degradation activity. After a week of daily treatment, the allergic conjunctivitis clinical scores in Dunkin-Hartley guinea pigs were evaluated. The guinea pigs were sensitized with intraperitoneal ovalbumin and then subjected to topical conjunctival challenge. Different doses of TiO2 NPs/C. officinalis (20 and 100 µg/kg), prednisolone (30 mg/kg), chlorpheniramine (10 mg/kg), or PBS (10 mL/kg) were administered. The levels of serum Immunoglobulins were measured by ELISA. It was observed that the levels of ovalbumin-specific IgE notably decreased. Notably, the treatment with TiO2 NPs/C. officinalis at both doses led to a notably lower clinical score for allergic conjunctivitis. Furthermore, the TiO2 NPs/C. officinalis-treated guinea pigs conjunctival tissue showed mild mononuclear infiltration compared with the intense conjunctival tissue inflammatory infiltration observed in the PBS-treated group. These data suggest that TiO2 NPs/C. officinalis may have clinical efficacy in alleviating allergic conjunctivitis.